Therapeutic derivatives of interleukin-22

ABSTRACT

The invention relates to novel derivatives of Interleukin-22 (IL-22), particularly those comprising a fatty acid covalently attached to an IL-22 protein, and their use in therapy.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 17/737,849, filed May 5, 2022 (allowed) which is a continuationof International Application No. PCT/EP2020/081523, filed on Nov. 9,2020, which claims priority to European Application No. 19207766.7 filedon Nov. 7, 2019. Each of these applications is incorporated herein byreference in its entirety.

REFERENCE TO THE ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(CTKI_001_D01US_SeqList_ST26.xml; Size: 36,083 bytes; and Date ofCreation: June 23, 2023) are herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to novel derivatives of Interleukin-22(IL-22), and in particular to derivatives comprising a fatty acidcovalently attached to an IL-22 protein. The invention also encompassesmethods for their production and their use in therapy, including thetreatment, prevention and amelioration of metabolic, liver, pulmonary,gut, kidney and skin diseases, disorders and conditions.

BACKGROUND OF THE INVENTION

IL-22 is a 146 amino acid protein with a molecular weight of 17 KDa. Itbelongs to the IL-10 family of cytokines and selectively activates aheterodimeric receptor consisting of an IL-receptor B subunit(IL-10RA2), which is ubiquitously expressed, and an IL-22 receptor Asubunit (IL-22RA1), which has an epithelial restricted expression. It isa unique cytokine in that it is released from immune cells, butselectively targets epithelial cells. Hence, the signaling pathwaysinduced by IL-22 may have relevance in different tissues (targetsinclude skin, intestine, lung, liver, kidney, pancreas and thymus), butIL-22 activates them in an epithelial-specific manner. A soluble bindingprotein, IL-22BP, neutralises IL-22 and thus regulates its effect.

IL-22 is released as a response to signals reflecting chemical ormechanical injury, e.g. aryl hydrocarbon receptor activation in responseto environmental toxins or tryptophan intermediates, and the activationof pattern recognition receptors, such as toll-like receptor 4, inresponse to proteins, fragments and debris from dying cells or invadingpathogens. IL-22 release is further stimulated by certain cytokines, inparticular IL-23 and to a lesser extent IL1β. IL-22 is thus secreted asa response to cues reflecting pathogen infection and immune activationtoo.

The effect of IL-22 is the result of an orchestrated engagement ofseveral activities/pathways. IL-22 acts on epithelial barrier tissuesand organs upon injury to protect the cells and maintain barrierfunction (e.g. through activation of anti-apoptotic gene programs). Italso accelerates repair (e.g. by inducing the proliferation of maturecells and activation of stem cells), prevents fibrosis (e.g. throughreducing epithelial-mesenchymal transition, antagonising the NLRP3inflammasome and inducing hepatic stellate cell senescence) and controlsinflammation (e.g. by inducing anti-microbial peptides and chemotaxissignals). IL-22 has been reported as able to treat a range of medicalconditions, including those often observed in diabetic or overweightmammals, such as hyperglycemia, hyperlipidemia and hyperinsulinemia.

However, IL-22 is generally cleared quickly from the body by thekidneys, which limits its use in clinical practice. Known methods forextending the half-life of circulating IL-22 therefore seek toartificially increase the size of IL-22 beyond 70 kDa, so as to avoidrenal clearance. Ligating IL-22 to an Fc antibody fragment is currentlythe best solution to this effect; Genentech and Generon Shanghai bothhave long-acting IL-22-Fc fusions in clinical development. ModifyingIL-22 with polyethylene glycol (PEGylation) is another known means foravoiding renal clearance.

However, these existing solutions are not without their disadvantages.The available data suggest that PEG itself is immunogenic andPEG-containing vacuoles are observed in cells with PEGylatedbiologicals. Decreased activity and heterogeneity are alsodisadvantageous aspects of PEGylation. Although Fc fusion technology isvery well known, adding an Fc antibody fragment represents a majorchange in the structure of IL-22, which affects its properties beyondhalf-life extension. As Fc fusion increases the size of the protein fromapproximately 17 kDa to approximately 85 kDa, properties such asdiffusion rate, distribution and receptor engagement kinetics may beaffected. For example, some Fc fusions are slowly absorbed and/or aretoo large for administration via certain routes. Both Genentech andGeneron also report moderate and reversible skin reactions asdose-limiting adverse effects of IL-22-Fc fusions. Furthermore, thepotency may be affected through steric hindrance caused by the largefusion partner.

A need therefore remains in the art for new biocompatible modifiers ofIL-22 that enhance circulating half-lives and demonstrate optimisedpharmacokinetic and pharmacodynamic properties compared to the nativemolecule. Ideally they should maintain potency and other properties ofthe native molecule and also avoid toxicity, immunogenicity and anyother adverse reactions demonstrated by known derivatives.

SUMMARY OF THE INVENTION

In a first aspect, there is provided a derivative of IL-22 comprising afatty acid covalently attached to an IL-22 protein.

In embodiments of the invention, the fatty acid is covalently attachedto the IL-22 protein by a linker.

The fatty acid may be of Formula I:

HOOC—(CH₂)_(x)—CO—*,

wherein x is an integer in the range of 10-18, optionally 12-18, 14-16or 16-18, and * designates a point of attachment to the IL-22 protein orlinker. It may be a fatty diacid, such as a C12, C14, C16, C18 or C20diacid. Advantageously, the fatty acid is a C16 or C18 diacid, and mostadvantageously it is a C18 diacid.

The IL-22 protein may be native mature human IL-22 (hereinafter“hIL-22”) or a variant thereof. The variant may be a substituted form ofhIL-22, optionally substituted at position 1, 21, 35, 64, 113 and/or114. It may comprise a substitution of hIL-22 selected from the groupconsisting of A1C, A1G, A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q,N64C, N64D, N64Q, N64W, Q113C, Q113R, K114C and K114R. Advantageously,the variant comprises a Cys residue at position 1 of hIL-22.

The variant may be an extended form of hIL-22. It may comprise anN-terminal peptide, such as an N-terminal trimer. Advantageously, thevariant comprises an N-terminal G-P-G.

The linker may comprise one or more amino acids, optionally includingglutamic acid (Glu) and/or lysine (Lys). The linker may include anoxyethylene glycine unit or multiple linked oxyethylene glycine units,optionally 2-5 such units, advantageously 2 units. The linker maycomprise one or more oligo(ethylene glycol) (OEG) residues. It maycomprise an ethylenediamine (C2DA) group and/or an acetamide (Ac) group.Advantageously, the linker comprises all of the aforementioned elementsin combination. In particular, the linker may be γGlu-OEG-OEG-C₂DA-Ac,γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc or γGlu-OEG-OEG-εLys-αAc.

The linker may be a Cys-reactive linker attached to a Cys residue in thehIL-22 or variant thereof. It may be attached at position −7, −5, 1, 6,33, 113, 114 or 153 of the hIL-22 or variant thereof (where positions−7, −5 etc. are as defined herein). As an example, the linker may beattached to a Cys residue substituted at position 1, 6, 33, 113 or 114of hIL-22. It may be attached to a Cys residue at position −5, −7 or 153relative to hIL-22. Advantageously, the linker is attached to a Cysresidue substituted at position 1 of hIL-22.

In an embodiment, the derivative comprises a C14, C16, C18 or C20 diacidcovalently attached by a linker to a variant of hIL-22, wherein thevariant comprises an N-terminal G-P-G and a Cys residue at position 1 ofhIL-22 and the linker is optionally attached to said Cys residue.Exemplary derivatives of the invention are those identified herein asDerivatives 1-10.

In a second aspect, there is provided a process for preparing aderivative of the first aspect comprising covalently attaching a fattyacid to an IL-22 protein.

In a third aspect, there is provided a pharmaceutical compositioncomprising a derivative of the first aspect, and a pharmaceuticallyacceptable vehicle.

In a fourth aspect, there is provided a derivative of the first aspector a pharmaceutical composition of the third aspect, for use in therapy.

In a fifth aspect, there is provided a derivative of the first aspect ora pharmaceutical composition of the third aspect, for use in a method oftreating a metabolic, liver, pulmonary, gut, kidney or skin disease,disorder or condition.

The metabolic disease, disorder or condition may be obesity, diabetestype 1, diabetes type 2, hyperlipidemia, hyperglycemia orhyperinsulinemia.

The liver disease, disorder or condition may be non-alcoholic fattyliver disease (NAFLD), non-alcoholic steatohepatitis (NASH), cirrhosis,alcoholic hepatitis, acute liver failure, chronic liver failure,acute-on-chronic liver failure (ACLF), acute liver injury, acetaminopheninduced liver toxicity, sclerosing cholangitis, biliary cirrhosis or apathological condition caused by surgery or transplantation.

The pulmonary disease, disorder or condition may be chronic obstructivepulmonary disease (COPD), cystic fibrosis, bronchiectasis, idiopathicpulmonary fibrosis, acute respiratory distress syndrome, a chemicalinjury, a viral infection, a bacterial infection or a fungal infection.

The gut disease, disorder or condition may be inflammatory bowel disease(IBD), ulcerative colitis, Crohn's disease, graft-versus-host-disease(GvHD), a chemical injury, a viral infection or a bacterial infection.

The kidney disease, disorder or condition may be acute kidney disease orchronic kidney disease.

The skin disease, disorder or condition may be a wound, inflammatorydisease or GvHD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate a (FIG. 1A) C18 diacid, (FIG. 1B) C16 diacid, and(FIG. 1C) C14 diacid, each connected to a linker comprising aCys-reactive unit. These combinations of fatty acids and linkers areemployed in the derivatives of the invention identified herein asDerivatives 1-10.

FIG. 2 illustrates the structure of a derivative of the inventionidentified herein as Derivative 1.

FIG. 3 illustrates the structure of a derivative of the inventionidentified herein as Derivative 6.

FIG. 4 illustrates the structure of a derivative of the inventionidentified herein as Derivative 10.

FIG. 5 illustrates the effect of daily dosing of hIL-22 and acomparative IL-22 variant having 10 backbone variations only (identifiedherein as Comparator 3) on blood glucose in an 8-day study in a diabetesmouse model (mean±SEM).

FIGS. 6A-6B illustrate the effect of daily dosing of a derivative of theinvention (herein identified as Derivative 1) compared to an IL-22-Fcfusion (specifically a human Fc N-terminally fused to hIL-22;hereinafter “hFc-hIL-22”) on (FIG. 6A) blood glucose, and (FIG. 6B) foodintake, in a 16-day study in a diabetes mouse model (mean±SEM; * meansp<0.05 using an unpaired t-test).

FIGS. 7A-7C illustrate the effect of daily dosing of Derivative 1 andhFc-hIL-22 on three different target engagement biomarkers, in a 16-daystudy in a diabetes mouse model (mean±SEM; *** means (A) p<0.0002, (B)p<0.0003 or (C) p<0.0026 using an unpaired t-test).

FIG. 8 illustrates a dose-response curve for daily dosing of aderivative of the invention (herein identified as Derivative 6) (threedifferent doses) compared to Derivative 1 and hFc-hIL-22 on bloodglucose in a 13-day study in a diabetes mouse model (mean±SEM).

FIGS. 9A-9B illustrate the effect of Derivatives 1 and 6 in preventingliver injury in an acetaminophen (APAP)-induced liver injury mousemodel, as evidenced by plasma levels of two different liver enzymes.Using Dunnett's test one-factor linear model, * means p<0.05 and **means p<0.01 compared to vehicle+APAP.

FIGS. 10A-10B illustrate the effect of Derivatives 1 and 6, (FIG. 10A)in preventing apoptosis and (FIG. 10B) on cellular proliferation, in anAPAP-induced liver injury mouse model. NS means non-significant.

FIGS. 11A-11C illustrate the effect of Derivative 6 in preventing and/orreducing (FIG. 11A) lung inflammation, and (FIG. 11B) and (FIG. 11C)lung fibrosis, in a bleomycin-induced lung injury rat model, compared toprednisolone.

FIG. 12 illustrates the effect of Derivative 6 in preventing coloninflammation in a dextran sulfate sodium (DSS)-induced colitis mousemodel. **** means p<0.0001 compared to vehicle (containing DSS).

FIG. 13 illustrates the effect of Derivative 6 compared to hFc-hIL-22 inpreventing mucosal epithelial wounding in the DSS-induced colitis mousemodel. Magnification 4×, scale bar=500 μm.

FIG. 14 illustrates plasma Regenerating Islet Derived Protein 3 Gamma(Reg3g) levels in the DSS-induced colitis mouse model, as a measure oftarget engagement (Reg3g is a target engagement marker of IL-22).

FIGS. 15A-15B illustrate the effect of Derivative 1 in preventing liverinjury in a Concanavalin A (ConA)-induced liver injury mouse model, asevidenced by serum levels of two different liver enzymes.

FIG. 16 illustrates the effect of Derivative 6 compared to hFc-hIL-22and the known fatty acid conjugated GLP-1 derivative, semaglutide, onbody weight in Diet Induced Obese mice.

DETAILED DESCRIPTION

In what follows, Greek letters are represented by their symbol ratherthan their written name. For example, α=alpha, ε=epsilon, γ=gamma andμ=mu. Amino acid residues may be identified by their full name,three-letter code or one-letter code, all of which are fully equivalent.

The term “derivative of IL-22”, as used herein, refers to an IL-22protein having a covalently attached fatty acid. The term encompassesboth derivatives in which the fatty acid is covalently attached to theIL-22 protein directly and those in which the covalent attachment is bya linker.

The covalent attachment of fatty acids is a proven technology forhalf-life extension of peptides and proteins and is a way of subtendinga fatty acid from the peptide or protein. It is known from marketedproducts for types 1 and 2 diabetes, such as insulins Levemir® (detemir)and Tresiba® (degludec), and glucagon-like peptide-1 (GLP-1) derivativesVictoza® (liraglutide) and Ozempic® (semaglutide).

Fatty acid attachment enables binding to albumin, thereby preventingrenal excretion and providing some steric protection againstproteolysis. Advantageously, it offers a minimal modification to IL-22compared to Fc fusion or PEGylation. In this regard, whilst Fc fusionand PEGylation aim to increase the size of IL-22 beyond the thresholdfor renal clearance, derivatives comprising a fatty acid covalentlyattached to an IL-22 protein retain a small size similar to that of theIL-22 protein. Thus, as the fatty acid attachment is a minimalmodification, the resultant derivative is believed to maintainnative-like properties including distribution, diffusion rate andreceptor engagement (binding, activation and trafficking) and minimiseimmunogenicity risk.

As above, fatty acid attachment has proven therapeutic efficacy ininsulin and GLP-1 derivatives for diabetes. However, IL-22 is a verydifferent protein in terms of its size, sequence and biologicalproperties. It was therefore counterintuitive to the inventors thatfatty acids could be covalently attached to IL-22 whilst maintainingtherapeutic effect. It was particularly surprising that such a minimalmodification to IL-22 could result in high potency (close to hIL-22)combined with a very long circulatory half-life.

In a first aspect, therefore, the invention relates to a derivative ofIL-22 comprising a fatty acid covalently attached to an IL-22 protein.The fatty acid may be covalently attached to the IL-22 protein directlyor via a linker, which itself can be devised of various subunits. Theterm, “IL-22 protein”, as used herein, can mean a native IL-22 protein,such as hIL-22, or a variant thereof. A “variant” can be a proteinhaving a similar amino acid sequence to that of the native protein, asfurther defined herein.

In nature, human IL-22 protein is synthesised with a signal peptide of33 amino acids for secretion. The mature human IL-22 protein (i.e.hIL-22) is 146 amino acids in length and has 80.8% sequence identitywith murine IL-22 (the latter being 147 amino acids in length). Theamino acid sequence of hIL-22 is identified herein as SEQ ID NO. 1. Likeother IL-10 family members, the IL-22 structure contains six α-helices(referred to as helices A to F).

The derivatives of the invention may thus have the native amino acidsequence of hIL-22. Alternatively, they may have one or more amino acidsequence variations within the native sequence. They may additionally oralternatively include one or more amino acid sequence variationsrelative to (i.e. outside) the native sequence. Thus, in an embodiment,the derivative comprises a fatty acid covalently attached to hIL-22 or avariant thereof.

Expressions such as “within”, “relative to”, “corresponding to” and“equivalent to” are used herein to characterise the site of changeand/or covalent attachment of a fatty acid in an IL-22 protein byreference to the sequence of the native protein, e.g. hIL-22. In SEQ IDNO. 1, the first amino acid residue of hIL-22 (alanine (Ala)) isassigned position 1.

Thus, a variation within the sequence of hIL-22 is a variation to any ofresidue numbers 1-146 in SEQ ID NO. 1. For example, a Glu substitutionfor the native Asp at residue 10 in hIL-22 is represented herein as“D10E”. If the derivative also has a fatty acid covalently attached atposition 10, it is herein referred to as attachment at residue “10E”.

A variation relative to the sequence of hIL-22, however, is a variationexternal to residue numbers 1-146 in SEQ ID NO. 1. For example,Derivative 2 as defined herein includes an N-terminal peptide of 15amino acids in length. The residues in the N-terminal peptide arenumbered negatively, starting from the residue attached to residue 1 inhIL-22, i.e. the first residue in the N-terminal peptide that isattached to residue 1 in hIL-22 is denoted “−1”. Thus, as Derivative 2has a fatty acid covalently attached at the 7^(th) residue in theN-terminal peptide starting from position −1 and this is Cys, thecovalent attachment site for Derivative 2 is herein referred to as“−7C”. Naturally, however, the numbering used in the sequence listingfor Derivative 2 starts from 1, in accordance with WIPO Standard ST.25;as such, position 1 in the sequence listing for Derivative 2 is actuallyresidue −7 as referred to herein.

Two, three, four, five or more variations may be made within the nativesequence to form the derivatives of the invention. For example, morethan 10, 15, 20, 25, 50, 75, 100 or even more than 125 variations may bemade in this regard. Any of residues 1-146 in the native sequence may bevaried. Exemplary residues for variation are residues 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 25, 26,27, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 44, 45, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 63, 64, 65, 67, 68, 69,70, 71, 72, 73, 74, 75, 77, 78, 79, 82, 83, 84, 86, 88, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 126, 127, 128, 129, 130, 132, 133, 134, 135, 137, 138, 139, 141,143, 144, 145 and/or 146 in hIL-22. Variation at residues 1, 21, 35, 64,113 and/or 114 is particularly advantageous.

The variations within the native sequence are typically amino acidsubstitutions. The term “substitution”, as used herein, can mean thereplacement of an amino acid in the native protein with another. Theymay be conservative or non-conservative substitutions. Exemplarysubstitutions are A1C, A1G, A1H, P2C, P2H, I3C, I3H, I3V, S4H, 54N, S5H,S5T, H6C, H6R, C7G, R8G, R8K, L9S, D10E, D105, K11C, K11G, K11V, S12C,N13C, N13G, F145, Q15C, Q15E, Q16V, P17L, Y18F, 119Q, T20V, N21C, N21D,N21Q, R22S, F24H, M25E, M25L, L26S, A27L, E29P, A30Q, L32C, L32R, A33C,A33N, D34F, N35C,

N35D, N35H, N35Q, N36Q, T37C, T37I, D38L, V39Q, R4OW, L41Q, I42P, E44R,K45A, F47T, H48G, H48R, G49N, V505, S51C, M52A, M52C, M52L, M52V, S53C,S53K, S53Y, E54D, E54F, R55Q, R55V, C56Q, L58K, M59I, Q61E, V62D, L63C,N64C, N64D, N64Q, N64W, F65G, L67Q, E69D, E69L, V705, L71C, F72D, F72L,P73C, P73L, Q74T, R77I, F78Q, Q79E, M82Y, Q83G, E84R, V86A, F88N, A90P,A90T, R91C, R91K, R91Y, L92R,

S93Y, N94C, N94Q, R95K, R95Q, L96E, S97K, T98C, T98N, T98S, C99V, H100S,E1025, G103D, D104Y, D105Y, L106E, L106Q, H107L, H107N, 1108L, Q109Y,R110C, R110K, N111K, V112E, Q113C, Q113R, K114C, K114R, L115V, K116Y,D117E, T118G, V119A, K120H, K121R, L122A, G123V, G126Y, E127C, I128V,K129V, G132Y, E133Q, L134P, D135M, L137D, F138R, M139L, M139R, L141Q,N143S, A144E, C145E, I146R and/or I146V. Advantageously, thesubstitution may be selected from the group consisting of A1C, A1G, A1H,N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W, Q113C,Q113R, K114C and K114R. Surprisingly, substitutions as employed in theinvention do not adversely affect IL-22 activity.

Particular combinations of substitutions include (i) A1G, N21D, N35D andN64D; (ii) A1G, I3V, S4N, S5T, H6R, R8K, D10E, K11V, T20V, H48R, M52A,S53K, E54D, R55Q, E69D, F72L, A90T, R91K, R95Q, T98S, E102S, L106Q,H107N, R110K, Q113R, K114R, D117E and I146V; (iii) A1G, I3V, S4N, S5T,H6R, R8K, D10E, K11V, T20V, H48R, M52A, S53K, E54D, R55Q, E69D, F72L,A90T, R91K, R95Q, T98S, E1025, L106Q, H107N, R110K, Q113R, K114R, D117Eand I146V; (iv) A1G, N35Q and N64Q; (v) A1G and N64C; (vi) A1G andQ113C; (vii) A1G and K114C; (viii) A1G and M25L; (ix) A1G and M52L; (x)A1G and M139L; (xi) A1G and N36Q; (xii) A1G and D117E; (xiii) A1G andN21Q; (xiv) A1G and N35Q; (xv) A1G and N64Q; (xvi) A1G, N21Q and N35Q;(xvii) A1G, N21Q and N64Q; (xviii) A1G, N21Q, N35Q and N64Q; (xix) A1Gand K11C; (xx) A1G and N13C; (xxi) N35Q and N64Q; (xxii) A1C, N35Q andN64Q; (xxiii) H6C, N35Q and N64Q; (xxiv) I3C, N35Q and N64Q; (xxv) P2C,N35Q and N64Q; (xxvi) L32C, N35Q and N64Q; (xxvii) N35Q, M52C and N64Q;(xxviii) N13C, N35Q and N64Q; (xxix) N21C, N35Q and N64Q; (xxx) N35Q,N64Q and N94C; (xxxi) N35Q, N64Q and P73C; (xxxii) N35Q, N64Q and Q113C;(xxxiii) N35Q, N64Q and R91C; (xxxiv) N35Q, N64Q and R110C; (xxxv) S12C,N35Q and N64Q; (xxxvi) N35Q, S51C and N64Q; (xxxvii) N35Q, S53C andN64Q; (xxxviii) N35Q, T37C and N64Q; (xxxix) N35Q, N64Q and T98C; (xxxx)Q15C, N35Q and N64Q; (xxxxi) N35C and N64Q; (xxxxii) H6C, N35Q and N64Q;(xxxxiii) A33C, N35Q and N64Q; and (xxxxiv) A1H, P2H, I3H, S4H, SSH,C7G, R8G, L9S, D105, K11G, N13G, F145, Q15E, Q16V, P17L, 18F, Y19Q,N21Q, R22S, F24H, M25E, L26S, A27L, E29P, A30Q, L32R, A33N, D34F, N35H,T37I, D38L, V39Q, R4OW, L41Q, I42P, E44R, K45A, F47T, H48G, G49N, V505,M52V, S53Y, E54F, R55V, C56Q, L58K, M59I, Q61E, V62D, L63C, N64W, F65G,L67Q, E69L, V70S, L71C, F72D, P73L, Q74T, R77I, F78Q, Q79E, M82Y, Q83G,E84R, V86A, F88N, A90P, R91Y, L92R, S93Y, N94Q, R95K, L96E, S97K, T98N,C99V, H100S, G103D, D104Y, D105Y, L106E, H107L, I108L, Q109Y, R111K,V112E, L115V, K116Y, D117E, T118G, V119A, K120H, K121R, L122A, G123V,G126Y, E127C, I128V, K129V, G132Y, E133Q, L134P, D135M, L137D, F138R,M139R, L141Q, N143S, A144E, C145E and I146R. Any and all combinations ofsubstitutions are envisaged and form part of the invention.

A derivative of the first aspect may typically comprise an amino acidsubstitution whereby Cys is substituted for a native residue, optionallyin any of the positions identified above, such as position 1, 2, 3, 6,11, 12, 13, 15, 21, 32, 33, 35, 37, 51, 52, 53, 63, 64, 71, 73, 91, 94,98, 110, 113, 114 and/or 127. Advantageously, the IL-22 protein includedin a derivative of the first aspect comprises a Cys residue at position1 of hIL-22. An A1C substitution combined with substitutions in twoglycosylation sites at positions 35 and 64 is particularly advantageous,as it leads to faster uptake without adversely affecting potency orhalf-life (see Derivatives 6 and 10 in Examples 1 and 2).

Alternatively, or in addition, the variations within the native sequencemay be amino acid insertions. Up to five, 10, 15, 20, 25, 30, 35, 40, 45or even up to 50 amino acids may be inserted within the native sequence.Trimers, pentamers, septamers, octamers, nonamers and 44-mers areparticularly advantageous in this regard. Exemplary sequences are shownin Table 1. Insertions can be made at any location in the nativesequence, but those in helix A (for example, at residue 30), loop CD(for example, at residue 75), helix D (for example, at residue 85)and/or helix F (for example, at residue 124) are preferred.

TABLE 1 Sequence of exemplary amino acid insertions SEQ n-merExemplary amino acid sequence ID NO. Trimer E-T-S n/a PentamerR-V-Q-F-Q or C-V-E-I-P 2, 3 Septamer G-S-G-S-G-S-C 4 OctamerI-E-A-L-T-P-H-S or Y-G-Q-R-Q-W- 5, 6 K-N Nonamer V-F-I-I-N-N-S-L-E 744-mer R-A-A-S-A-G-S-Y-S-E-W-S-M-T-P- 8 R-F-T-P-W-W-E-T-K-I-D-P-P-V-M-N-I-T-Q-V-N-G-S-L-L-V-I-L-H

Sequence variations relative to the amino acid sequence of hIL-22, ifpresent, typically include an extension, such as the addition of apeptide at the N-terminal end. The peptide may consist of up to five,10, 15, 20, 25, 30, 35, 40, 45 or even up to 50 amino acids. Monomers,trimers, octamers, 13-mers, 15-mers, 16-mers, 21-mers, 28-mers areparticularly advantageous in this regard. Exemplary sequences are shownin Table 2. Suitably, the IL-22 protein included in a derivative of thefirst aspect comprises an N-terminal G-P-G. In a particularly preferredexample, the derivative of the first aspect comprises both a Cys residueat position 1 of hIL-22 (SEQ ID NO. 1) and an N-terminal G-P-G. This hasbeen found to create a derivative with a very good half-life and potency(see Derivatives 1, 3 and 5 in Examples 1 and 2).

TABLE 2 Sequence of exemplary N-terminal peptides SEQ n-merExemplary amino acid sequence ID NO. Monomer C, G or M n/a Trimer G-P-Gn/a Octamer G-P-A-C-E-P-E-E  9 13-mer G-G-S-S-G-S-G-S-E-V-L-F-Q 1015-mer G-P-G-S-G-S-G-S-C-G-S-G-S-G-S 11 16-merG-G-S-S-G-S-G-S-E-V-L-F-Q-G-P-G 12 21-merG-G-S-S-G-S-G-S-E-V-L-F-Q-G-P-A- 13 C-E-P-E-E 28-merG-G-S-S-G-S-G-S-E-V-L-F-Q-G-P-G- 14 S-G-S-G-S-C-G-S-G-S-G-SSequence variations relative to the amino acid sequence of hIL-22, ifpresent, may include the addition of a peptide at the C-terminal end.The peptide may consist of up to five, 10, 20, 25, 30, 35, 40, 45 oreven up to 50 amino acids. A septamer is particularly advantageous inthis regard, optionally having the amino acid sequence, G-S-G-S-G-S-C(SEQ ID NO. 15).

The derivatives of the invention may include both an N-terminal and aC-terminal peptide in addition to the native or variant hIL-22 aminoacid sequence as herein described. Any combination of the N- andC-terminal peptides described herein is envisaged and expressly includedin the invention.

It will be appreciated that the invention extends to any derivative ofIL-22, which comprises a fatty acid covalently attached to hIL-22 or avariant thereof. The “variant” can be a protein having at least 10%sequence identity with hIL-22. In an embodiment, the variant has atleast 20%, or even at least 30%, sequence identity with hIL-22. Thevariant may have “substantially the amino acid sequence” of hIL-22,which can mean a sequence that has at least 40% sequence identity withthe amino acid sequence of hIL-22. Accordingly, in an embodiment, aderivative of the first aspect has at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95% or 100% amino acidsequence identity with hIL-22. Exemplary IL-22 protein variants, whichare incorporated in the particular derivatives of the inventiondisclosed in the experimental section, are set forth in SEQ ID NOs.16-21.

The skilled technician will appreciate how to calculate the percentageidentity between two amino acid sequences. An alignment of the twosequences must first be prepared, followed by calculation of thesequence identity value. The percentage identity for two sequences maytake different values depending on: (i) the method used to align thesequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman(implemented in different programs), or structural alignment from 3Dcomparison; and (ii) the parameters used by the alignment method, forexample, local versus global alignment, the pair-score matrix used (forexample, BLOSUM62, PAM250, Gonnet etc.) and gap-penalty, for example,functional form and constants.

Having made the alignment, there are many different ways of calculatingpercentage identity between the two sequences. For example, one maydivide the number of identities by: (i) the length of shortest sequence;(ii) the length of alignment; (iii) the mean length of sequence; (iv)the number of non-gap positions; or (iv) the number of equivalencedpositions excluding overhangs. Furthermore, it will be appreciated thatpercentage identity is also strongly length-dependent. Therefore, theshorter a pair of sequences is, the higher the sequence identity one mayexpect to occur by chance.

Hence, it will be appreciated that the accurate alignment of amino acidsequences is a complex process. The popular multiple alignment programClustalW [48,49] is a preferred way for generating multiple alignmentsof proteins in accordance with the invention. Suitable parameters forClustalW may be as follows: For protein alignments: Gap OpenPenalty=10.0, Gap Extension Penalty=0.2, and Matrix=Gonnet. For DNA andProtein alignments: ENDGAP=−1, and GAPDIST=4. Those skilled in the artwill be aware that it may be necessary to vary these and otherparameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino acidsequences may then be calculated from such an alignment as (N/T)*100,where N is the number of positions at which the sequences share anidentical residue, and T is the total number of positions comparedincluding gaps but excluding overhangs. Hence, a most preferred methodfor calculating percentage identity between two sequences comprises (i)preparing a sequence alignment using the ClustalW program using asuitable set of parameters, for example, as set out above; and (ii)inserting the values of N and T into the following formula: SequenceIdentity=(N/T)*100.

Alternative methods for identifying similar sequences will be known tothose skilled in the art.

Suitably, a derivative of the first aspect comprises 200 amino acids orless. For example, the derivative comprises less than 190, less than180, less than 170, less than 160 or even less than 150 amino acids.Suitably, the derivative will comprise at least 146 amino acids,however, this being the number of amino acids in hIL-22. It may compriseat least 150 amino acids, at least 160 amino acids, at least 170 aminoacids or even at least 180 amino acids. The derivatives of the inventioncan comprise proteins of any length within the above ranges, but theywill typically be 146-180 amino acids in length.

The derivatives of the invention, whether having the native or a variantamino acid sequence, include a fatty acid covalently attached to theIL-22 protein. The fatty acid is typically covalently attached to theIL-22 protein by a linker. The fatty acid and linker are suitablyconnected to each other via an amide bond, and the linker is covalentlyattached to the IL-22 protein. The fatty acid and linker may thus bepresent as a side chain on the IL-22 protein. It was surprising to theinventors that a covalently attached fatty acid does not adverselyaffect IL-22 activity. It was particularly surprising that fatty acidattachment is associated with additional advantages, such asprolongation of half-life.

The fatty acid may be any suitable fatty acid. In particular, the fattyacid may be of Formula I:

HOOC—(CH₂)_(x)—CO—*,

wherein x is an integer in the range of 10-18, optionally 12-18, 14-16or 16-18, and * designates a point of attachment to the IL-22 protein orlinker. It may be a fatty diacid, such as a C12, C14, C16, C18 or C20diacid. Advantageously, the fatty acid is a C16 or C18 diacid, and mostadvantageously it is a C18 diacid.

For example, —(CH₂)_(x)— in Formula I may be a straight alkylene inwhich x is 10. This fatty acid may be conveniently referred to as C12diacid, i.e. a fatty di-carboxylic acid with 12 carbon atoms.Alternatively, —(CH₂)_(x)— in Formula I may be a straight alkylene inwhich x is 12. This fatty acid may be conveniently referred to as C14diacid, i.e. a fatty di-carboxylic acid with 14 carbon atoms. In asimilar fashion, —(CH₂)_(x)— in Formula I may be a straight alkylene inwhich x is 14 (C16 diacid), 16 (C18 diacid) or 18 (C20 diacid).Suitably, a derivative of the first aspect includes a C14, C16, C18 orC20 diacid; more suitably, a C16 or C18 diacid, and even more suitably aC18 diacid.

The diacid may be capable of forming non-covalent associations withalbumin, thereby promoting circulation of the derivative in the bloodstream. The shorter diacids (e.g. C16 diacid) have lower albuminaffinity and thus a shorter half-life than the longer diacids (e.g. C18diacid). However, they are still long acting derivatives with anexpected half-life in man of over one day.

Fatty acid attachment will, in itself, also stabilise the IL-22 proteinagainst proteolytic degradation. The resulting half-life is typicallysimilar to that of IL-22-Fc fusions (i.e. greatly improved compared tohIL-22).

The derivatives of the first aspect may comprise particular combinationsof a fatty acid and IL-22 protein. For example, a C14, C16, C18 or C20diacid may be attached to an IL-22 protein comprising a Cys residue atposition 1 of hIL-22 and/or an N-terminal G-P-G. In one example, aderivative of the first aspect comprises a C18 diacid and the IL-22protein comprises both a Cys residue at position 1 of hIL-22 and anN-terminal G-P-G.

As above, the fatty acid is suitably connected to a linker, which isattached to the IL-22 protein. The linker may comprise several linkerelements, including one or more amino acids such as one or more Gluand/or Lys residues. The linker may include an oxyethylene glycine unitor multiple linked oxyethylene glycine units, optionally 2-5 such units,advantageously 2 units. One or more OEG residues, C₂DA and/or Ac groupsmay alternatively or additionally be included. The linker may comprise aCys-reactive unit. A “Cys-reactive unit”, as used herein, can mean afunctional unit that is able to react with the sulphur atom of a Cys tocreate a carbon-sulphur covalent bond. The Cys-reactive unit can haveany of several forms, but suitably includes a carbon atom attached to aleaving group, which leaving group becomes displaced by the sulphur atomof the Cys during formation of the carbon-sulphur bond. The leavinggroup may be a halogen, optionally a bromine atom. This bromide leavinggroup can be alpha to an actamide functional group; advantageously it isa bromo-acetamide functional group. The leaving group may alternativelybe a functionalised hydroxyl group of the form mesylate or tosylate, oran unfunctionalised hydroxyl group. Further, the leaving group can be amaleimide or other functional group. Exemplary linkers includeγGlu-OEG-OEG-C₂DA-Ac, γGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc andγGlu-OEG-OEG-εLys-αAc, but any suitable linker may be employed.

The fatty acid, or linker, may be attached to any amino acid residue inthe IL-22 protein. Exemplary in this regard are residues −7, −5, 1, 6,33, 113, 114 and 153 in or relative to the hIL-22 amino acid sequence.The native residue is typically substituted with Cys or Lys to enableattachment of the fatty acid or linker. Alternatively the fatty acid orlinker can be attached at a native Cys or Lys residue. Suitably, thefatty acid or linker is attached to a Cys residue substituted atposition 1, 6, 33, 113 or 114 of hIL-22 or to a Cys residue at position−5, −7 or 153 relative to hIL-22. In particular, the fatty acid orlinker may be attached to a Cys residue substituted at position 1 ofhIL-22.

The attachment of the fatty acid or linker to the IL-22 protein is acovalent attachment. For example, a Cys-reactive fatty acid or linkermay be used to attach the fatty acid or linker to a Cys residue in theIL-22 protein. The fatty acid or linker may be covalently attached tothe sulphur atom of the Cys residue via a thioether bond. Alternatively,a Lys-reactive fatty acid or linker may be used to attach the fatty acidor linker to a Lys residue in the IL-22 protein. The fatty acid orlinker may alternatively be covalently attached to the free amine (—NH₂)group in the N-terminus of the IL-22 protein (irrespective of the aminoacid in position 1). Attachment can proceed as with Cys attachment,albeit with sub-stoichiometric amounts of fatty acid or linkercontaining a suitable N-reactive species. The fatty acid or linker maybe presented in the form of an aldehyde (the N-reactive species) and becovalently attached to the free amine employing a classically knownreductive amination.

A derivative of the first aspect thus suitably comprises a C14, C16, C18or C20 diacid attached by a linker to a variant of hIL-22, wherein thevariant comprises an N-terminal G-P-G and a Cys residue at position 1 ofhIL-22 and the linker is optionally attached to the Cys residue.

Exemplary derivatives of the first aspect comprise an IL-22 protein asset forth in any of SEQ ID NOs. 16-21. Particularly advantageousderivatives are shown in Table 3, illustrated in FIGS. 1-4 andexemplified herein.

TABLE 3 Exemplary derivatives of IL-22 Covalent SEQ attachment Fatty IDSequence variations ID NO. site Linker acid Derivative 1A1C substitution & G- 16  1C γGlu-OEG- C18 P-G OEG-C₂DA- diacidN-terminal peptide Ac Derivative 2 G-P-G-S-G-S-G-S-C-G- 17 −7C γGlu-OEG-C18 S-G-S-G-S OEG-C₂DA- diacid N-terminal peptide Ac Derivative 3A1C substitution & G- 16  1C γGlu-OEG- C16 P-G OEG-C₂DA- diacidN-terminal peptide Ac Derivative 4 G-P-G-S-G-S-G-S-C-G- 17 −7C γGlu-OEG-C16 S-G-S-G-S OEG-C₂DA- diacid N-terminal peptide Ac Derivative 5A1C substitution & G- 16  1C γGlu-γGlu- C14 P-G γGlu-γGlu- diacidN-terminal peptide OEG-OEG- ϵLys-αAc Derivative 6 A1C, N35Q, N64Q 18  1CγGlu-OEG- C18 substitutions & G-P-G OEG-C₂DA- diacid N-terminal peptideAc Derivative 7 A1C, N35Q, N64Q 19  1C γGlu-OEG- C18 substitutionsOEG-C₂DA- diacid Ac Derivative 8 H6C, N35Q, N64Q 20  6C γGlu-OEG- C18substitutions & G OEG-C₂DA- diacid N-terminal peptide Ac Derivative 9A33C, N35Q, N64Q 21 33C γGlu-OEG- C18 substitutions OEG-C₂DA- diacid AcDerivative 10 A1C, N35Q, N64Q 18  1C γGlu-OEG- C16 substitutions & G-P-GOEG-C₂DA- diacid N-terminal peptide Ac

FIG. 1A illustrates a C18 diacid connected to a linker comprising aCys-reactive unit. This is the fatty acid and linker (side chain) usedin Derivatives 1, 2 and 6-9. FIG. 1B illustrates a C16 diacid connectedto a linker comprising a Cys-reactive unit. This is the fatty acid and 5linker (side chain) used in Derivatives 3, 4 and 10. FIG. 1C illustratesa C14 diacid connected to a linker comprising a Cys-reactive unit. Thisis the fatty acid and linker (side chain) used in Derivative 5.

Derivatives 1, 6 and 10 are illustrated in FIGS. 2-4 , respectively.

The derivatives of the invention may exist in different stereoisomericforms and the invention relates to all of these.

According to a second aspect of the invention, there is provided aprocess for preparing a derivative of the first aspect comprisingcovalently attaching a fatty acid to an IL-22 protein.

The process may be used to produce any of the different derivatives ofIL-22 described or envisaged herein, but it is particularly advantageouswhen a fatty acid is covalently attached to a variant IL-22 protein.Thus, in an embodiment, the IL-22 protein employed in the second aspectis a substituted form of hIL-22, optionally substituted at position 1,21, 35, 64, 113 and/or 114. Exemplary substitutions include A1C, A1G,A1H, N21C, N21D, N21Q, N35C, N35D, N35H, N35Q, N64C, N64D, N64Q, N64W,Q113C, Q113R, K114C and/or K114R. Preferably, the IL-22 protein issubstituted with a Cys residue at position 1.

The fatty acid can be obtained by any means known in the art, includingrecombinant means. Suitable fatty acids are commercially available orreadily derived from available starting materials using standardchemical synthesis.

The IL-22 protein can be obtained by any means known in the art,including recombinant means. The production of recombinant hIL-22 hasbeen previously described and is well-known in the art. Desired variantIL-22 proteins can be produced in a similar manner. An experiencedinvestigator in the field would be readily able to identify suitablenucleic acid sequences that encode the desired variant IL-22 proteins.The skilled person would hence be readily able to execute this part ofthe invention, based upon the existing knowledge in the art. Suitably,the IL-22 proteins are produced in mammalian systems, such as in Chinesehamster ovary (CHO) cells, using standard techniques. A polyhistidinetag (His-tag) may be employed to aid affinity purification of therecombinant proteins.

In this regard, IL-22 proteins as used in the invention can be preparedusing a post expression cleavable His-tag—an N- or C-terminal additionof less than 10, preferably six, histidine residues that can be purifiedby affinity to a nickel column. The His-tag is linked to the N- orC-terminal of the protein via a linker that can be digested by a knownprotease to leave the free IL-22 protein. The cleavable His-tag can havethe amino acid sequence, HEIHHHHGGSSGSGSEVLFQ (SEQ ID NO. 25), and theprotease-cleavable linker can be a tobacco etch virus (TEV) linker,whose consensus sequence for the native cut sites is ENLYFQ\S (SEQ IDNO. 26), where ‘\’ denotes the cleaved peptide bond or a humanrhinovirus-14 3C (HRV14-3C) protease cleavable linker with EVLFQconsensus cleavage site. Cleavage may be achieved by incubatingapproximately 10 μg protease with 2.5 protein and 10 mM2-mercaptoethanol at room temperature for 4 h.

To further illustrate the invention, a representative process forprotein preparation is provided as follows. The process involvespreparing a plasmid DNA that encodes the desired amino acid sequence ofthe IL-22 protein. This plasmid can be transiently transfected into acell line, for example CHO-K1, which is allowed to grow in a relevantmedium before growth is increased by the addition of a known enhancer.The secreted IL-22 protein can then be harvested through known methodsof centrifugation and sterile filtration before the protein is purifiedon a nickel column. Following concentration and buffer exchange theHis-tag is removed using a HRV14-3C protease before alkylation with afatty acid (described further below) and final purification and bufferexchange. Analysis of the final product using SDS-PAGE, size exclusionchromatography or liquid chromatography with tandem mass spectrometry(LC-MS-MS) with, or without, deglycosylation can be used to ensure thequality of the final product.

The fatty acid can be covalently attached to the IL-22 protein eitherdirectly or using a linker as described for the first aspect. The linkercan be obtained by any means known in the art. A representative methodfor preparing the fatty acid and linker, if employed, is as follows(exemplified by the C16 diacid used in Derivative 10, but any derivativecould be made using a similar method).

A solution of N-(benzyloxycarbonyloxy) succinimide (100 g, 401 mmol) indichloromethane (500 ml) is added to a solution of ethylene diamine (189ml, 2.81 mol) in dichloromethane (750 ml). After 30 minutes thesuspension is filtered, washed and concentrated in vacuo. The residue isdiluted with toluene (750 ml), washed and extracted with dichloromethane(4×200 ml), dried over anhydrous sodium sulphate, filtered, concentratedin vacuo and diluted with hexanes (200 ml). A 4 M solution of hydrogenchloride in ether (100 ml, 400 mmol) is added to the solution, theresulting suspension concentrated in vacuo and diluted with hexanes (1l). The precipitated solid is filtered, washed with hexanes and dried invacuo to give (2-aminoethyl) carbamic acid benzyl ester hydrochloride asa white powder.

2-Chlorotrityl resin 100-200 is loaded with{2-[2-(9H-fluoren-9-ylmethoxycarbonylamino)-ethoxy]-ethoxy}-acetic acid(Fmoc-Ado-OH, 17.5 g, 45.4 mmol) The Fmoc group is removed and asolution of 0-6-chloro-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate (TCTU, 24.2 g, 68.1 mmol) andN,N-diisopropylethylamine (21.4 ml, 123 mmol) in N,N dimethylformamide(140 ml) is added to the resin and the mixture shaken for one hour. Theresin is filtered and washed. The Fmoc group is removed by treatmentwith 20% piperidine as before. The resin is washed as before.

A solution of (S)-2-(fluoren-9-ylmethoxycarbonylamino)-pentanedioic acid1-tert-butyl ester (Fmoc-Glu-OtBu, 29.0 g, 68.1 mmol), TCTU (24.2 g,68.1 mmol) and N,N-diisopropylethylamine (21.4 ml, 123 mmol) in N,Ndimethylformamide (140 ml) is added to the resin and the mixture shakenfor one hour. The resin is filtered and washed as before. The Fmoc groupis removed by treatment with 20% piperidine as before. The resin iswashed as before.

A solution of 16-tert-butoxy)-16-oxohexadecanoic acid (23.3 g, 68.1mmol), TCTU (24.2 g, 68.1 mmol) and N,N diisopropylethylamine (21.4 ml,123 mmol) in N,N-dimethylformamide/dichloromethane mixture (4:1, 200 ml)is added to the resin. The resin is shaken for one hour, filtered andwashed with N,N-dimethylformamide (3×250 ml), dichloromethane (2×250ml), methanol (2×250 ml) and dichloromethane (6×250 ml). The product iscleaved from the resin by treatment with 2,2,2-trifluoroethanol (250 ml)for 18 hours. The resin is filtered off and washed with dichloromethane(2×250 ml), 2-propanol/dichloromethane mixture (1:1, 2×250 ml),2-propanol (250 ml) and dichloromethane (3×250 ml).

The solutions are combined, the solvent is evaporated and crude productpurified by flash column chromatography. Pure(S)-22-(tert-butoxycarbonyl)-41,41-dimethyl-10,19,24,39-tetraoxo-3,6,12,15,40-pentaoxa-9,18,23-triazadotetracontanoicacid is dried in vacuo and obtained as a pale yellow thick yellow oil.

2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 11.4 g, 30.1 mmol) and triethylamine (8.77ml, 62.9 mmol) are subsequently added to a solution of(S)-22-(tert-butoxycarbonyl)-41,41-dimethyl-10,19,24,39-tetraoxo-3,6,12,15,40-pentaoxa-9,18,23-triazadotetracontanoicacid (22.4 g, 27.4 mmol) in dry dichloromethane (110 ml). Triethylamine(72 ml, 41.0 mmol) is added to a suspension of (2-amino-ethyl)-carbamicacid benzyl ester hydrochloride (6.94 g, 30.1 mmol) in drydichloromethane (165 ml) and the resulting mixture is added to the abovesolution. The mixture is stirred at room temperature overnight and thenevaporated to dryness. The residue is re-dissolved and washed; driedover anhydrous sodium sulphate and evaporated column chromatography(Silicagel 60, 0.040-0.060 mm; eluent: dichloromethane/methanol 95:5) toafford15-[(S)3-(2-{2-[(2-{2-[(2-benzyloxycarbonylamino-ethylcarbamoyl)-methoxy]-ethoxy}ethyl-carbamoyl)methoxy]ethoxy)-ethylcarbamoyl)-1-tert-butoxycarbonylpropylcarbamoyl]-pentadecanoicacid tert-butyl ester as a pale yellow thick oil.

Palladium on carbon (10%, 1.27 g, 1.20 mmol) is added to a solution ofthe above compound (23.8 g, 24.0 mmol) in methanol (350 ml) and theresulting mixture hydrogenated at normal pressure for four hours. Thecatalyst is filtered off and the filtrate evaporated to dryness. Theresidue is evaporated several times from dichloromethane in order toremove residues of methanol and dried in vacuo to yieldtert-butyl(S)-1-amino-25-tert-butoxycarbonyl)-4,13,22,27-tetraoxo-6,9,15,18-tetraoxa-3,12,21,26-tetraazadotetracontan-42-oateas a thick colourless oil.

N,N-Diisopropylethylamine (4.98 ml, 28.6 mmol) is added to a solution ofthe above amine (20.5 g, 23.8 mmol) in dry dichloromethane (290 ml) at−30° C. under argon. Bromoacetyl bromide (2.48 ml, 28.6 mmol) is addeddropwise and the resulting solution is stirred at −30° C. for anadditional three hours. The cooling bath is removed, the mixture isstirred at room temperature for one hour, and the solvent is removed invacuo. The residue is re-dissolved in ethyl acetate (450 ml) and washedwith 5% aqueous solution of citric acid (300 ml). The phases areseparated within one hour. The organic layer is left to separateovernight to give three phases. The clear aqueous layer is removed andthe residual two phases shaken with a saturated aqueous solution ofpotassium bromide (100 ml). The phases are left to separate overnight,the aqueous phase removed and the organic phase dried over anhydroussodium sulphate. The solvent is removed in vacuo and the residuepurified by flash column chromatography: dichloromethane/methanol 95:5)to affordtertbutyl(S)-1-bromo-28-tert-butoxycarbonyl)-2,7,16,25,30-pentaoxo-9,12,18,21-tetraoxa-3,6,15,24,29-pentaazapenta-tetracontan-45-oateas a colourless solid.

The above compound (19.5 g, 19.8 mmol) is dissolved in trifluoroaceticacid (120 ml) and the resulting solution is stirred at room temperaturefor 1.5 hours. Trifluoroacetic acid is removed in vacuo and the residueis evaporated from dichloromethane (6×200 ml). Diethyl ether (200 ml) isadded to the oily residue and the mixture stirred overnight to give asuspension. The solid product is filtered, washed with diethyl ether andhexanes and dried in vacuo to afford the desired product15-{(S)-1-carboxy3-[2-(2-{[2-(2-{[2-(2-Bromoacetylamino)ethylcarbamoyl]methoxy}-ethoxyethyl-carbamoyl]methoxy}ethoxylethylcarbamoyl]propylcarbamoyl}pentadecanoicacid as a white powder.

Covalent attachment of the fatty acid or linker to the IL-22 protein maybe carried out using standard procedures in the art. The linker, ifemployed, thus enables covalent attachment of the IL-22 protein to thefatty acid. By way of a non-limiting example, a Cys-reactive fatty acidor linker may be reacted with the sulphur atom of a Cys residue in theIL-22 protein, so forming a thioether bond. Suitable conditions for thecovalent attachment step may be exemplified as follows: Tris in water isadded to IL-22 protein (70 mg) in Tris and NaCl-buffer (1.35 mg/ml), toadjust to pH 8. Bis(p-sulfonatophenyl)- phenylphosphine dihydratedipotassium (BSPP) salt (12 mg), dissolved in water, is added andstirred gently for four hours at room temperature.15-{(5)-1-Carboxy-3-[2-(2-{[2-2-[2-(2-bromoacetylamino)-ethylcarbamoyl]ethoxy}ethoxy)ethylcarbamoyl]methoxylethoxy)ethylcarbamoyl]propyl-carbamoyl}pentadecanoicacid (19 mg, 0.022 mmol) in ethanol (0.5 ml) is added and the mixturestirred gently overnight. MiliQ water (150 ml) is added to lower theconductivity to 2.5 mS/cm. The mixture is then purified using anionexchange on a MonoQ 10/100 GL column using binding buffer (20 mM Tris,pH 8.0), elution buffer (20 mM Tris, 500 mM NaCl, pH 8.0), flow 6 ml anda gradient of 0-80% elution buffer over 60 column volumes.

The derivatives of the invention may be purified using any suitableprocedure known in the art, such as chromatography, electrophoresis,differential solubility or extraction.

As described herein, the inventors were surprised to find that fattyacids could be covalently attached to an IL-22 protein whilstmaintaining biological activity. It was particularly surprising thatsuch a minimal modification to IL-22 could result in high potency (closeto hIL-22) combined with a very long circulatory half-life. Thisparticular combination of properties may be highly desirable.

The potency of the derivatives may be determined in an in vitro assaywith whole cells expressing human IL-22 receptors. For example, theresponse of the human IL-22 receptors may be measured using baby hamsterkidney (BHK) cells overexpressing IL-22R1, IL-10R2 and a phospho-STAT3(pSTAT3) responsive reporter gene. Alternatively, HepG2 cellsendogenously expressing the IL-22 receptor may be used. Activation ofthe receptors leads to activation of the STAT3 signaling pathway, whichcan be measured using a luciferase reporter gene with a STAT3-inducedpromoter or by assaying pSTAT3, for example. Non-limiting examples ofsuch assays are described in Example 2. In vivo potency may bedetermined in animal models or in clinical trials, as is known in theart.

The half maximal effective concentration (EC₅₀) value is often used as ameasure of the potency of a drug. As this represents the concentrationof drug required to produce 50% of the maximal effect, the lower theEC₅₀ value, the better the potency. The derivatives of the inventionsuitably have a potency (EC₅₀ value) measured using IL-22receptor-mediated STAT3 activation in cells of below 1.5 nM, below 1.25nM, below 1 nM, below 0.75 nM, below 0.5 nM, below 0.25 nM or even below0.1 nM (e.g. determined as described in Example 2). The derivatives ofthe invention suitably have a potency (EC₅₀ value) measured by assayingpSTAT3 in cells of below 15 nM, below 12 nM, below 10 nM, below 7 nM oreven below 5 nM (e.g. determined as described in Example 2).

Advantageously, the potency of the derivatives of IL-22 may be higherthan that of IL-22-Fc fusions. For example, Genentech has reported a34-fold reduction in in vitro potency for its IL-22-Fc fusion,UTTR1147A, compared to hIL-22 (Stefanich et al., Biochem Pharmacol,2018, 152:224-235). By contrast, covalent attachment of a fatty acid tohIL-22 has been shown to cause only a seven-fold reduction in potency(see Derivative 1 in the Examples). Whilst both IL-22-Fc fusions and thederivatives of the present invention may be comparable in terms of theirimproved half-life over hIL-22 and biological function in at least somesettings, the derivatives of the invention may have the additionaladvantage of minimal loss of potency.

The circulatory elimination half-life (T_(1/2)) of the derivatives maybe determined in vivo by administering the derivatives subcutaneously orintravenously in a suitable animal model, such as a mouse, rat orminipig. Suitable methods are described in Example 1. By way of anon-limiting example, the derivatives of the first aspect have acirculatory half-life after subcutaneous or intravenous administrationto mice of at least one hour, at least three hours, at least five hoursor even at least eight hours. The derivatives may have a circulatoryhalf-life after subcutaneous or intravenous administration to rats of atleast three hours, at least five hours, at least eight hours, at least10 hours or even at least 13 hours. The derivatives may have acirculatory half-life after subcutaneous or intravenous administrationto minipigs of at least 25 hours, at least 40 hours, at least 70 hoursor even at least 100 hours (all determined, e.g. as described in Example1).

As exemplified herein, the inventors have also found that thederivatives of the invention are absorbed rapidly in vivo.Advantageously, absorption of the derivatives may occur faster than thatof IL-22-Fc fusions. Mean absorption time is an accurate parameter formeasuring uptake because it is independent of dose and maximum plasmaconcentration following drug administration. It can be calculated basedupon mean residence time, i.e. the time that a drug spends in the bodyprior to elimination once absorption has been completed. The derivativesof the invention suitably have a mean absorption time of below 100 h,below 90 h, below 80 h, below 70 h or even below 60 h (e.g. determinedas described in Example 1).

The derivatives of the invention also have good biophysical properties,such as high physical stability and/or solubility, which may be measuredusing standard methods in the art.

Therefore, according to a third aspect of the invention, there isprovided a pharmaceutical composition comprising a derivative of thefirst aspect and a pharmaceutically acceptable vehicle.

A pharmaceutical composition of the third aspect may comprise any of thedifferent derivatives of IL-22 described or envisaged herein. Suitably,it comprises one of the derivatives of IL-22 identified herein asDerivative 1-10.

A derivative of the first aspect, or a pharmaceutical composition of thethird aspect, will suitably demonstrate increased circulatoryelimination half-life compared to hIL-22. Advantageously it willdemonstrate increased circulatory elimination half-life compared tohIL-22 by at least 50%, at least 75%, at least 100% or more.

The pharmaceutical compositions of the third aspect may be prepared bycombining a therapeutically effective amount of a derivative of thefirst aspect with a pharmaceutically acceptable vehicle. The formulationof pharmaceutically active ingredients with various excipients is knownin the art.

A “therapeutically effective amount” of a derivative of the first aspectis any amount which, when administered to a subject, is the amount ofderivative that is needed to treat the disease, disorder or condition orproduce the desired effect.

For example, the therapeutically effective amount of derivative used maybe from about mg to about 1000 mg, and preferably from about 0.01 mg toabout 500 mg. It is preferred that the amount of derivative is an amountfrom about 0.1 mg to about 100 mg, and most preferably from about 0.5 mgto about 50 mg. As a guide, the dose of derivative used in the mice inExample 3 described herein was 0.5 mg/kg (administered subcutaneously).

A “pharmaceutically acceptable vehicle” as referred to herein, is anyknown compound or combination of known compounds that are known to thoseskilled in the art to be useful in formulating pharmaceuticalcompositions.

In one embodiment, the pharmaceutically acceptable vehicle may be asolid; optionally the composition may be in the form of a powder forresuspension. A solid pharmaceutically acceptable vehicle may includeone or more substances which may also act as flavouring agents,lubricants, solubilisers, suspending agents, dyes, fillers, glidants,inert binders, preservatives or dyes. The vehicle may also be anencapsulating material. In powders, the vehicle is a finely dividedsolid that is in admixture with the finely divided derivatives accordingto the invention. The powders preferably contain up to 99% derivative.Suitable solid vehicles include, for example calcium phosphate,magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin,cellulose and ion exchange resins.

In another embodiment, the pharmaceutical vehicle may be a gel and thecomposition may be in the form of a cream or the like.

However, the pharmaceutical vehicle may be a liquid; optionally thepharmaceutical composition is in the form of a solution. Liquid vehiclesare used in preparing solutions, suspensions, emulsions, syrups, elixirsand pressurised compositions. The derivative according to the inventionmay be dissolved or suspended in a pharmaceutically acceptable liquidvehicle such as water, an organic solvent, a mixture of both orpharmaceutically acceptable oils or fats. The liquid vehicle can containother suitable pharmaceutical additives such as solubilisers,emulsifiers, buffers, preservatives, sweeteners, flavouring agents,suspending agents, thickening agents, colours, viscosity regulators,stabilisers or osmo-regulators. Suitable examples of liquid vehicles forparenteral administration include water (partially containing additivesas above, for example, cellulose derivatives, preferably sodiumcarboxymethyl cellulose solution), alcohols (including monohydricalcohols and polyhydric alcohols, for example, glycols) and theirderivatives, and oils (for example, fractionated coconut oil and arachisoil). For parenteral administration, the vehicle can also be an oilyester such as ethyl oleate and isopropyl myristate. Sterile liquidvehicles are useful in sterile liquid form compositions for parenteraladministration. The liquid vehicle for pressurised compositions can be ahalogenated hydrocarbon or other pharmaceutically acceptable propellant.

The process for preparing a pharmaceutical composition of the inventionmay thus comprise the usual steps that are standard in the art.

According to a fourth aspect of the invention, therefore, there isprovided a derivative of the first aspect, or a pharmaceuticalcomposition of the third aspect, for use in therapy. A method oftreating a subject with a derivative of the invention, or apharmaceutical composition comprising the same, is also provided. Any ofthe different derivatives of IL-22 described or envisaged herein areexpressly included in these aspects of the invention.

Terms such as “treating” and “therapy”, as used herein, expresslyinclude the treatment, amelioration or prevention of a disease, disorderor condition.

The derivative of IL-22 or pharmaceutical composition comprising thesame may be administered directly into a subject to be treated. Thederivative or pharmaceutical composition may be administered by anymeans, including by inhalation, by injection, topically or ocularly.When administered by inhalation, it may be via the nose or the mouth.Preferably, the derivative or pharmaceutical composition is administeredby injection, typically subcutaneously or intravenously. The derivativestherefore have a clear advantage over Fc fusions in their flexibility ofadministration (e.g. by injection, by inhalation, topical application orocular delivery) because of their smaller size and higher potency. Itwill be appreciated that administration, into a subject to be treated,of a derivative of the invention will result in the increasedcirculation time compared to hIL-22, and that this will aide in treatinga disease, disorder or condition. As above, ‘treating’ also includesameliorating and preventing a disease, disorder or condition.

Liquid pharmaceutical compositions, which are sterile solutions orsuspensions, can be utilised by, for example, intramuscular,intrathecal, epidural, intraperitoneal and particularly subcutaneous orintravenous injection. The derivative may be prepared as a sterile solidcomposition that may be dissolved or suspended at the time ofadministration using sterile water, saline or other appropriate sterileinjectable medium.

Forms useful for inhalation include sterile solutions, emulsions andsuspensions. Alternatively the derivatives may be administered in theform of a fine powder or aerosol via a Dischaler® or Turbohaler®. Nasalinhalations may suitably be in the form of a fine powder or aerosolnasal spray or modified Dischaler® or Turbohaler®.

Topical formulations include solutions, creams, foams, gels, lotions,ointments, pastes, tinctures and powders. They may be epicutaneous, i.e.applied directly to the skin, or applied to mucous membranes.

Formulations for ocular administration are typically solutions,suspensions and ointments for topical application, e.g. in the form ofeye drops. Alternatively sterile solutions or suspensions can beutilised by intraocular injection. The derivative may be prepared as asterile solid composition that may be dissolved or suspended at the timeof administration using sterile water, saline or other appropriatesterile injectable medium. The formulation may be for subconjunctival,intravitreal, retrobulbar or intracameral injection.

A derivative or pharmaceutical composition of the invention may beadministered to any subject in need thereof. A “subject”, as usedherein, may be a vertebrate, mammal or domestic animal. Hence,derivatives and compositions according to the invention may be used totreat any mammal, for example livestock (for example, a horse), pets, ormay be used in other veterinary applications. Most preferably, thesubject is a human being. The derivatives and compositions need not onlybe administered to those already showing signs of a disease, disorder orcondition. Rather, they can be administered to apparently healthysubjects as a purely preventative measure against the possibility ofsuch a disease, disorder or condition in future.

It will be appreciated that derivatives of IL-22 and compositionsaccording to the invention may be used in a monotherapy (i.e. the soleuse of that derivative or composition), for treating a disease, disorderor condition. Alternatively, derivatives and compositions according tothe invention may be used as an adjunct to, or in combination with,known therapies for treating a disease, disorder or condition.

It will be appreciated that the amount of the derivative of IL-22 thatis required is determined by its biological activity, half-life andbioavailability, which in turn depends on the mode of administration,the physiochemical properties of the derivative and composition, andwhether it is being used as a monotherapy or in a combined therapy. Thefrequency of administration will also be influenced by the half-life ofthe derivative within the subject being treated. Optimal dosages to beadministered may be determined by those skilled in the art, and willvary with the particular derivative in use, the strength of thepharmaceutical composition, the mode of administration, and theadvancement of the disease, disorder or condition. Additional factorsdepending on the particular subject being treated will result in a needto adjust dosages, including subject age, weight, gender, diet and timeof administration.

Generally, a daily dose of between 0.001 μg/kg of body weight and 10mg/kg of body weight of derivative of IL-22 according to the inventionmay be used for treating a disease, disorder or condition, dependingupon which derivative or composition is used. More preferably, the dailydose is between 0.01 μg/kg of body weight and 1 mg/kg of body weight,more preferably between 0.1 μg/kg and 500 μg/kg body weight, and mostpreferably between approximately 0.1 μg/kg and 100 μg/kg body weight.

The derivative of IL-22 or composition may be administered before,during or after onset of the disease, disorder or condition. Daily dosesmay be given as a single administration (for example, a single dailyinjection). Alternatively, the derivative or composition may requireadministration twice or more times during a day. As an example,derivatives may be administered as two (or more depending upon theseverity of the disease, disorder or condition being treated) dailydoses of between 0.07 μg and 700 mg (i.e. assuming a body weight of 70kg). A patient receiving treatment may take a first dose upon waking andthen a second dose in the evening (if on a two-dose regime) or at 3- or4-hourly intervals thereafter. Doses may alternatively be given once aweek, every fortnight or once a month, or more frequently, for example,two or three times weekly. Known procedures, such as thoseconventionally employed by the pharmaceutical industry (for example, invivo experimentation, clinical trials, etc.), may be used to formspecific formulations of the derivatives and compositions according tothe invention and precise therapeutic regimes (such as daily doses ofthe agents and the frequency of administration).

Many studies have demonstrated key effects of IL-22 in multipleepithelial injury models in especially lung, liver, intestine, kidney,skin, pancreas and thymus. Mechanistically, several pathways within e.g.anti-apoptosis, proliferation, innate immunity, anti-oxidative stress,anti-fibrosis, and stem cell/progenitor cell recruitment have been welldocumented to meditate IL-22 effects in studies by multipleinvestigators. Key mechanistic findings have been further confirmed invitro using human cell lines or in human ex vivo models (e.g. primaryhuman intestinal organoids). The strong role of IL-22 in preventing celldeath, securing regeneration, and controlling inflammation in epithelialinjury is therefore well established.

Many studies are performed by analysing genetic models (IL-22 knock-outor transgenic overexpression) subjected to injury. In these studies, thelack of IL-22 or overexpression of IL-22 will be there at the time ofinjury. In other studies, IL-22 is neutralised with antibodies at thetime of injury and, in some cases, IL-22 is neutralised beyond the acuteinjury phase (e.g. sub-acutely or well into a regeneration phase). Otherstudies move closer to a treatment scenario by looking at effects ofexogenously administered IL-22. Important to note, when lookingholistically at the available literature, is that the different models,whether knock-out, overexpression, IL-22 neutralisation before or afterinjury or exogenous protein dosing, paint the same picture of IL-22protecting the injured organs and driving regeneration. This indicates abroad application and a broad time window for IL-22 treatment potential,and also shows why a longer-acting IL-22 protein than hIL-22 isrequired.

Thus, according to a fifth aspect of the invention, there is provided aderivative of the first aspect, or a pharmaceutical composition of thethird aspect, for use in a method of treating a metabolic, liver,pulmonary, gut, kidney or skin disease, disorder or condition. Any ofthe different derivatives of IL-22 described or envisaged herein areexpressly included in this aspect of the invention.

The metabolic disease, disorder or condition may be obesity, diabetestype 1, diabetes type 2, hyperlipidemia, hyperglycemia orhyperinsulinemia.

The liver disease, disorder or condition may be NAFLD, NASH, cirrhosis,alcoholic hepatitis, acute liver failure, chronic liver failure, ACLF,acetaminophen induced liver toxicity, acute liver injury, sclerosingcholangitis, biliary cirrhosis or a pathological condition caused bysurgery or transplantation.

The pulmonary disease, disorder or condition may be COPD, cysticfibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acuterespiratory distress syndrome, a chemical injury, a viral infection, abacterial infection or a fungal infection.

The gut disease, disorder or condition may be IBD, ulcerative colitis,Crohn's disease, GvHD, a chemical injury, a viral infection or abacterial infection.

The kidney disease, disorder or condition may be acute kidney disease orchronic kidney disease.

The skin disease, disorder or condition may be a wound, inflammatorydisease or GvHD.

A method of treating a subject having a condition responsive to IL-22treatment, such as one or more of the above diseases, disorders orconditions, with a derivative of IL-22, or a pharmaceutical compositioncomprising the same, is also provided.

The derivative of IL-22 has all of the features specified for the firstaspect of the invention. The pharmaceutical composition has all of thefeatures specified for the third aspect of the invention. The method oftreating a subject having a condition responsive to IL-22 treatment,such as one or more of the above diseases, disorders or conditions, hasall of the features specified for the fourth aspect of the invention.

There is no restriction on which derivative of IL-22 or composition asdescribed herein should be administered to which patient. Rather, it isintended that any of the derivatives and compositions described hereincan be administered to any patient as described herein.

All of the features described herein (including any accompanying claims,abstract and drawings), and/or all of the steps of any method or processso disclosed, may be combined with any of the above aspects in anycombination, except combinations where at least some of such featuresand/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodimentsof the same may be carried into effect, reference will now be made tothe Examples, which are not intended to limit the invention in any way.

EXAMPLES

The materials and methods employed in the studies described in theExamples were as follows, unless where otherwise indicated.

Derivatives

Table 4 provides an overview of the derivatives of IL-22 and comparatorsrepresented in the data sets.

The derivatives of IL-22 had various backbones, types of fatty acid andsites of covalent attachment and were thus representative of thediversity of the derivatives encompassed by the invention. The linkerused in all cases was γGlu-OEG-OEG-C2DA-Ac. In all cases the linker wasattached to residue 1C, except for Derivatives 2 (−7C), 4 (−7C), 8 (6C)and 9 (33 C). Whilst Derivative 7 exemplifies covalent attachment at 1C,it lacks the G-P-G N-terminal peptide present in all of the otherderivatives that have a fatty acid covalently attached at 1C.

The comparators were hIL-22, hFc-hIL-22 (a recombinant fusion protein)and hIL-22 variants (i.e. hIL-22 having one or more backbone variationsonly).

TABLE 4Overview of key derivatives and comparators represented in data setsSEQ ID NO. Fatty acid or ID (for IL-22 protein) Backbone variationother protractor hIL-22 SEQ ID NO. 1 None None hFc-hIL-22 SEQ ID NO. 1None Fc Comparator 1 SEQ ID NO. 22 A1G None Comparator 2 SEQ ID NO. 23A1G, N21D, N35D, N64D None Comparator 3 SEQ ID NO. 24 A1G, N35Q, N64QNone Comparator 4 SEQ ID NO. 16 G-P-G N-terminal None peptide, A1CComparator 5 SEQ ID NO. 18 G-P-G N-terminal None peptide, A1C, N35Q,N64Q Derivative 1 SEQ ID NO. 16 G-P-G N-terminal C18 diacid peptide, A1CDerivative 2 SEQ ID NO. 17 G-P-G-S-G-S-G-S-C-G- C18 diacid S-G-S-G-SN-terminal peptide Derivative 3 SEQ ID NO. 16 G-P-G N-terminalC16 diacid peptide, A1C Derivative 4 SEQ ID NO. 17 G-P-G-S-G-S-G-S-C-G-C16 diacid S-G-S-G-S N-terminal peptide Derivative 6 SEQ ID NO. 18G-P-G N-terminal C18 diacid peptide, A1C, N35Q, N64Q Derivative 7SEQ ID NO. 19 A1C, N35Q, N64Q C18 diacid Derivative 8 SEQ ID NO. 20G N-terminal peptide, C18 diacid H6C, N35Q, N64Q Derivative 9SEQ ID NO. 21 A33C, N35Q, N64Q C18 diacid Derivative 10 SEQ ID NO. 18G-P-G N-terminal C16 diacid peptide, A1C, N35Q, N64Q

A quality control analysis of the derivatives produced for the exampleswas carried out as follows.

Intact mass of proteins was determined in a post-deglycosylated sampleby adding 20 μl of a 1 mg/ml sample to 2 μl N-Glycosidase F at roomtemperature for 48 h. The samples were then diluted to 0.2 mg/ml withPBS at pH 7.4, and analysed using a Synapt G2 connected to Waters SynaptG2, with Waters MassLynx 4.1. A 10-90 Column Acquity UPLC Protein BEH C41.7 μm 1×100 mm was used with the following mobile phase(s): A: 0.1%formic acid in water; and B: acetonitrile, 0.09% formic acid. Flow wasat 120 μ1/min, UV 214 nm (20 pts/s) and the gradient was as shown inTable 5.

TABLE 5 Gradient (% and min) employed for quality control of derivativesof IL-22 Time Flow % (min) (ml/min) A Initial 0.12 90 1 0.12 90 17 0.1210 18 0.12 0 19 0.12 0 20 0.12 90 25 0.12 90

The results are shown in Table 6.

TABLE 6 Mass and retention time observed for key derivatives of IL-22Deglycosylated Observed Retention theoretical deglycosylated time IDmass (Da) mass (Da) (min) Derivative 17807.6 17807.0 13.81 1 Derivative17779.6 17778.5 11.68 3 Derivative 18658.4 18657.5 7.11 4 Derivative18225.0 18224.0 7.28 5 Derivative 17832.7 17832.0 7.40 6

The quality control data thus verified that the intended derivatives hadindeed been produced.

The following are exemplified protocols, merely intended to illustratethe claimed invention. The exact number of animals and time coursesemployed in the studies can be varied, as would be known to a personskilled in the art.

Example 1—Pharmacokinetic Study Methods

Pharmacokinetic studies were carried out on selected derivatives in mice(n=27), rats (n=4-8) and minipigs (n=2-5). The derivatives of IL-22 weretested alongside hIL-22, hFc-hIL-22 and/or hIL-22 variants ascomparators.

(i) Mice & Rats

8-week old C57B1/6 male mice and five Sprague Dawley male rats wereobtained from Taconic Biosciences. The mice were housed in groups of 10.Animals were acclimatised for one week prior to the experiments. Bodyweight was measured prior to dosing, which is important forpharmacokinetic calculations. The animals were awake throughout theexperiment, with access to food and water.

All derivatives and comparators were prepared as 0.3 mg/ml solutions inPBS, pH 7.4, for use in mice and 0.5 mg/ml solutions for use in rats. Adose of 2.0 mg/kg was tested in mice. A dose of 1 mg/kg was tested inrats.

The derivatives and comparators were administered to the animalssubcutaneously. Blood samples were taken at specific time points afterdosing.

Sparse sampling was used in mice; thus, 27 mice were dosed with aderivative of IL-22 or comparator and blood samples were taken fromthree different mice at each of the following time points: 5 min, 15min, 30 min, 45 min, 60 min, 75 min, 90 min, 105 min, 120 min, 150 min,3 h, 4 h, 6 h, 8 h, 16 h, 24 h, 32 h and 48 h. Each mouse therefore hadjust two samples taken during the course of the study. After the lastsample was taken, the mice were euthanised by cervical dislocation.

Five rats were dosed with a derivative of IL-22 or comparator and threeblood samples were taken at each of the following time points: 5 min, 15min, 30 min, 45 min, 60 min, 75 min, min, 105 min, 120 min, 150 min, 3h, 4 h, 6 h, 8 h and 24 h. Each rat had 17 samples taken during thecourse of the study. After the last sample was taken, the rats wereeuthanised by carbon dioxide.

Blood samples (100 μl) were taken from mice and rats by tongue blood andtransferred to EDTA tubes (Microvette® VetMed 200 K3E, Sarstedt nr09.1293.100). The blood was centrifuged for five minutes at 8000 G, 4°C. within 20 minutes of being drawn. The plasma samples (40-5011.1) weretransferred to half micronic tubes.

(ii) Minipigs

9-month old female Gottingen minipigs having a body weight ofapproximately 15 kg were obtained from Ellegaard Gottingen Minipigs A/S.An acclimatisation period of approximately 18 days was allowed beforesurgery (insertion of catheters), during which time the minipigs weresocialised and trained for subcutaneous dosing and blood sampling fromthe catheter. Three to five days before surgery the minipigs weresingle-housed. Six days before dosing all minipigs had two centralvenous catheters (Cook Medical, C-TPNS-6.5-90-REDO, silicon, size 6.5french, 106 cm long type TPN) inserted, which allowed a recovery timeafter surgery of at least five days before study start (dosing).

All derivatives and comparators were prepared as solutions in PBS, pH7.4. The doses used were 0.1 mg/kg (administered intravenously) or 0.2mg/kg (administered subcutaneously).

Minipigs were lightly anaesthetized with Propofol during the dosing.Intravenous injections were administered to minipigs through the longcentral catheter. After administration, the catheter was flushed with 10ml sterile saline. Subcutaneous injection was given in 5 mm depth usinga 25 G needle. The needle was kept in the skin for 10 s after injectionto avoid back flow.

Blood samples were taken from the minipigs at the following time pointsafter intravenous dosing: 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24h, 28 h, 48 h, 72 h, 96 h, 144 h, 168 h, 192 h, 216 h, 240 h, 264 h, 312h, 336 h, 360 h, 384 h, 408 h, 432 h and 480 h. Blood samples were takenat the following time points after subcutaneous dosing: 1.5 h, 2 h, 3 h,4 h, 5 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26h, 28 h, 46 h, 52 h, 72 h, 96 h, 144 h, 168 h, 192 h, 216 h, 240 h, 264h, 312 h, 336 h, 360 h, 384 h, 408 h, 432 h and 480 h.

Blood samples (1 ml) were collected from minipigs in EDTA tubes (1.3 mltube containing K3EDTA to yield 1.6 mg K3EDTA/ml blood (Sarstedt,Germany)). Samples were kept on wet ice for a maximum of 30 min untilcentrifugation (10 min, 4° C., 2000 G). 20011.1 plasma was transferredinto Micronic tubes for measurement of the derivatives of IL-22 orcomparators and stored at −20° C. until analysis.

(iii) Sample Processing

Plasma levels of derivatives of IL-22 or comparators were measured usingin-house developed luminescent oxygen channeling (LOCI®) assays aspreviously described (Poulsen et al. J Biomol Screen, 2007,12(2):240-7). During the assays, a concentration-dependentbead-analyte-immune complex was created, resulting in light output,which was measured on a Perkin Elmer Envision reader. Coupling ofantibodies to beads, biotinylation of antibodies and LOCI assayprocedure were performed as previously described (Petersen et al., JPharmaceut Biomed, 2010, 51(1):217-24). Calibrators and quality control(QC) samples were produced in the same matrix as the study samples.Assay precision (% CV) was assessed and shown to be lower than 20% forall the tested samples.

The assay used anti-human IL-22 monoclonal antibody (R&D SystemsMAB7822)-conjugated acceptor beads together with biotinylated monoclonalantibody (R&D Systems BAM7821; raised against human IL-22) and genericstreptavidin-coated donor beads. The lower limit of quantification(LLOQ) for human IL-22 in rat plasma was 4 pM. Each derivative orcomparator was, however, measured against a calibrator row of the samederivative. The cross-reactivity of each derivative or comparatoragainst hIL-22 was measured and used to adjust the assay sensitivity.

Plasma concentration-time profiles were measured for minipigs using anon-compartmental analysis (NCA) in Phoenix WinNonlin Professional 6.4(Pharsight Inc). Calculations were performed using individualconcentrations, weighting by 1/(Y*Y), and using linear log trapezoidal.Intravenous dosing was used because circulatory elimination half-life(T_(1/2)) was the primary screening parameter. Clearance and volume ofdistribution were secondary parameters of interest, hence the reason forfrequent blood samples during day 1 of the study.

The sole parameter measured to assess pharmacokinetics in mice and ratswas circulatory elimination half-life (T_(1/2)). In minipigs, theadditional parameters measured were maximum (peak) plasma concentrationfollowing drug administration (C_(max)), time to reach C_(max)(T_(max)), area under the plasma drug concentration-time curve (AUC;which reflects the actual body exposure to drug after administration ofa dose of the drug) normalised for drug dose (AUC/D), mean residencetime (MRT; i.e. the time that the drug spends in the body prior toelimination once absorption has been completed), mean absorption time(MAT) and systemic availability of the administrated dose (i.e.bioavailability; F). MAT is calculated as MRT following subcutaneousadministration (MRTsc) minus MRT following intravenous administration(MRTiv).

Results

Table 7 shows the results obtained in mice, Table 8 shows the resultsobtained in rats and Tables 9 and 10 show the results obtained inminipigs. ND=not determined. IV=intravenous administration.SC=subcutaneous administration.

TABLE 7 Pharmacokinetic data obtained in mice Route of T_(1/2) IDAdministration (h) hFC-hIL-22 IV ND SC 30.0 Comparator 1 IV 0.4 SC 1.4Comparator 3 IV 0.4 SC 0.7 Derivative 1 IV 6.0 SC 8.3 Derivative 6 IV6.6 SC 9.1

As shown in Table 7, hIL-22 variants having backbone variations only(Comparators 1 and 3) had a short circulatory half-life, regardless ofthe route of administration. Protraction with Fc fusion (hFc-hIL-22)considerably increased half-life. Covalent attachment of fatty acid (C18diacid; Derivatives 1 and 6) resulted in an intermediary circulatoryhalf-life in mice. The derivatives of IL-22 circulated for longer inmice when administered subcutaneously compared to intravenously.

TABLE 8 Pharmacokinetic data obtained in rats Route of T_(1/2) IDAdministration (h) Comparator 1 IV 0.2 SC ND Derivative 1 IV 11.1 SC10.9 Derivative 3 IV 4.3 SC 9.7 Derivative 6 IV 10.8 SC 12.3

As shown in Table 8, the hIL-22 variant having a backbone variation only(Comparator 1) had a short circulatory half-life. Covalent attachment offatty acid (Derivatives 1, 3 and 6) resulted in an increased circulatoryhalf-life in rats, regardless of the fatty acid (C16 vs C18 diacid)employed and route of administration. The derivatives of IL-22 typicallycirculated for longer when administered subcutaneously compared tointravenously.

TABLE 9 Pharmacokinetic data obtained in minipigs Administration T_(1/2)ID route (h) hIL-22 IV 4.6 SC 6.6 hFc-hIL-22 IV 65.4 SC 141.0 Comparator1 IV 3.9 SC 8.7 Comparator 2 IV 3.5 SC 3.7 Derivative 1 IV 53.9 SC NDDerivative 6 IV 66.5 SC 106.0 Derivative 10 IV ND SC 40.0

As shown in Table 9, the hIL-22 variants having backbone variations only(Comparators 1 and 2) had a short circulatory half-life, comparable withhIL-22. The comparator Fc fusion (hFc-hIL-22) and all of the derivativesof IL-22 (Derivatives 1, 6 and 10) had a significantly increasedcirculating half-life. The derivatives of IL-22 had a circulatinghalf-life of over 50 hours in minipig when administered intravenously,which was on par with the comparator IL-22-Fc fusion.

TABLE 10 Pharmacokinetic data obtained in minipigs AUC/D AdministrationCmax Tmax (h*kg*pmol/ MRT MAT F ID route (nmol/l) (h) l/pmol) (h) (h)(%) hFc-hIL- IV 56.0 0.05 407 91.7 ND ND 22 SC 14.8 5.00 365 192.0 10089.7 Derivative IV 98.9 0.05 551 85.6 ND ND  6 SC 37.6 8.00 399 146 60.372.4

As shown in Table 10, a faster MAT was demonstrated for the derivativeof IL-22 (Derivative 6) compared to the comparator Fc fusion(hFc-IL-22). MAT is a more precise measure of drug uptake than simplycomparing T_(max), as it also takes into consideration differences inC_(max) (T_(max) is influenced by both dose and C_(max)). Minipigs wereused for this study, rather than mice or rats, because of theirsimilarity to humans.

Conclusion

The known fatty acid alkylated GLP-1 derivative, semaglutide, has ahalf-life of 46 hours in minipig (Lau et al., J Med Chem, 2015,58(18):7370-80) and a half-life of 160 hours in man, corresponding to aonce-weekly dosing profile with a peak to trough ratio of 2. Thehalf-life of the Fc-fusion GLP-1 derivative, dulaglutide, is similar.

The half-life demonstrated by the derivatives of IL-22 in minipig, of atleast 40 hours when administered subcutaneously and over 50 hours whenadministered intravenously, is therefore assumed to correspond to aonce-weekly dosing profile in man with a peak to trough ratio of 2.

The data hence show that the derivatives of the invention enhancedcirculating half-lives of IL-22 and demonstrated optimisedpharmacokinetic and pharmacodynamic properties, so offering a new andimproved treatment for a diverse range of indications, includingmetabolic, liver, pulmonary, gut, kidney, eye, thymus, pancreas, andskin diseases, disorders and conditions.

Example 2—In Vitro Potency Study Methods

Two in vitro assays were employed to study potency.

The first was a reporter gene assay in BHK cells, which had been tripletransfected with IL-22Ra, IL-10Rb and a luciferase with STAT3-inducedpromoter. This is a highly sensitive, high-throughput assay, whichmeasured IL-22 receptor-mediated STAT3 activation.

A stable reporter BHK cell line was generated using the followingplasmids: (i) hIL-10Rb in pcDNA3,1hygro(+), (ii) IL22R inpcDNA3,1(Zeocin) and (iii) 2xKZdel2 in pGL4.20. The cell line henceexpressed the human IL-10Rb, human IL-22Ra and luciferase reporter undercontrol of a pSTAT3 driven promoter.

On Day 0 of the assay protocol, the cells were seeded in basal media(for 500 ml: DMEM+Glutamax (Gibco, cat. no.: 31966-021), 10% (w/v) fetalcalf serum (FCS; contains albumin) (50 ml) and 1% (w/v)penicillin-streptomycin (P/S) (5 ml)) at 15,000-20,000 cells/well in a96-well plate (Corning #3842, black, clear bottom). On Day 1, the mediawas removed by inverting the plate. Fresh basal media was added, at 50μl per well, and the cells were incubated for 60 minutes.

The derivatives of IL-22 were tested alongside hIL-22 and hIL-22variants having backbone variations only as comparators. The ‘n’ numberof animals used to test each derivative or comparator ranged from 1-36.

Thus, 50 μl of a diluted derivative or comparator (diluted in basalmedia) was added to each well and the plate left for four hours. Thederivatives and comparators were therefore 2× diluted, as they werediluted into the 50 μl media already in the wells. The stimulation wasended after four hours by adding 100 μl Steadylite plus reagent (PerkinElmer cat no. 6066759). The plate was sealed with TopSeal A, shaken at450 rpm for 15 minutes, then read using Mithras or a similar system nolater than after 12 hours.

Data analysis was performed using Graphpad Prism. The half maximaleffective concentration (EC₅₀) of each derivative or comparator wasassessed as a measure of its potency. EC₅₀ was determined usingLog(compound) vs response—variable slope (4p). Hill slope wasconstrained to 1 as standard.

The second in vitro potency assay measured pSTAT3 in HepG2 cells—a humanliver-derived cell line endogenously expressing IL-22Ra and IL-10Rb.

On Day 1, HepG2 Cells were plated at 25,000-30,000 cells/well in a96-well plate (Biocoat #35-4407 Becton Dickinson). The cell media usedfor plating and passaging was DMEM(1×)+25 mM (4.5 g/l) glucose,-pyruvate (Gibco, cat. no. 61965-026)+10% (w/v) FCS+1% (w/v) P/S. On Day2, the cells were ready for assay. The cells were starved with 0.1%(w/v) FCS (i.e. a very low albumin concentration) in DMEM (Gibco, cat.no. 61965-026)−50 μl was added to each well and left for 60 minutes.

Tests were performed in seven concentrations of each derivative orcomparator as standard (0.001, 0.01, 0.1, 1, 10, 100, 1000 nM) usingtechnical duplicates. Thus, 50 μl of a diluted derivative or comparator(diluted in 0.1% (w/v) FCS in DMEM) was added to each well and the plateleft for 15 minutes. The derivatives and comparators were therefore 2×diluted, as they were diluted into the 50 μl media already in the wells.To lyse the cells, media was removed from the cells and 50 μl of freshlyprepared 1× lysis buffer (SureFire lysis buffer from kit) was added toeach well. The plate was agitated at 350 rpm for 10 minutes at roomtemperature.

The AlphaScreen® SureFire® STAT3 (p-Tyr705) assay protocol (Perkin Elmercat.no. TGRS3S (500-10K-50K)) was followed to measure IL-22 inducedphosphorylation of STAT3. In this regard, 4 μl of lysate was transferredto a 384-well proxiplate for assay (adding 4 μl of positive and negativecontrol). Immediately prior to use, Acceptor mix was prepared (bydiluting Activation buffer 5-fold in reaction buffer and dilutingAcceptor beads fold in the diluted buffer). 5 μl of Acceptor mix wasadded to each well, the plate sealed with Topseal A adhesive film andincubated for two hours at room temperature. Immediately prior to use,Donor mix was prepared (by diluting Donor beads 20-fold in Dilutionbuffer). 2 μl of donor mix was added to the wells under subdued light.The plate was again sealed with Topseal A adhesive film and incubatedfor two hours at room temperature. The plate was read on an AlphaTechnology-compatible plate reader.

Data analysis was performed using Graphpad Prism. First, a non-linearregression was conducted using Log(compound) vs. response−variable slope(4p) analysis in Prism. Hill slope was constrained to 1. The Y=top fromthe control compound (either His tagged hIL-22 or hIL-22) was then usedfor a normalisation in Prism. 0% was set to the smallest value in eachdata set and 100% to Y=top from the above non-linear regression (for thecontrol). A non-linear regression was repeated as above and %activity/wt and ECso of the tested derivatives read in the results undertop and ECso respectively.

Results

Table 11 shows the ECso of key derivatives and comparators measured inthe BHK cell reporter gene assay for IL-22 receptor mediated STAT3activation.

TABLE 11 EC₅₀ values for key derivatives and comparators in BHK cellassay EC₅₀ ID (nM) hIL-22 0.07 Comparator 1 0.06 Comparator 5 0.19Comparator 14 0.09 Derivative 1 0.48 Derivative 3 0.30 Derivative 4 0.18Derivative 6 0.61 Derivative 7 0.09 Derivative 8 1.24 Derivative 9 0.28Derivative 10 0.37

As the BHK cell assay contained high amounts of albumin, the measuredEC₅₀ incorporated the effect of albumin binding when testing thederivatives.

Comparator 4, an IL-22 variant having backbone variations only, wasshown to be equipotent to hIL-22. Derivative 3, having the same backboneas Comparator 4, but covalently attached to a medium affinity albuminbinder (C16 diacid), exhibited a four-fold reduction in potency comparedto hIL-22. Derivative 1, again having the same backbone, but covalentlyattached to a high affinity albumin binder (C18 diacid), exhibited onlya seven-fold reduction in potency compared to hIL-22.

A scan of alkylation positions and backbone variations has beenperformed with off-set in a 35Q, 64Q background (i.e. two of the threeIL-22 glycosylation sites mutated) by comparing the results forDerivatives 6-9. The covalent attachment sites in these derivatives hadbeen chosen based on an analysis of the IL-22 structure identifyingpositions that are expected to be surface exposed and not involved inreceptor binding. The results obtained with these derivativesdemonstrate that Cys substitution and fatty acid covalent attachment maybe tolerated in several (select) positions, which was surprising to theinventors.

Table 12 shows the EC₅₀ of key derivatives and comparators measured inthe HepG2 cell assay for pSTAT3.

TABLE 12 EC₅₀ values for key derivatives and comparators in HepG2 cellassay EC₅₀ ID (nM) hIL-22 3.88 Comparator 1 4.73 Comparator 4 12.11Derivative 1 10.13 Derivative 2 6.98 Derivative 6 14.86

In the HepG2 cell assay with endogenous expression level of receptors,little signal amplification and no albumin, Derivative 1 had a 2.5-foldreduced potency compared to hIL-22 (similar to Comparator 4, an hIL-22variant having the same backbone as Derivative 1, but no fatty acid).

Table 13 collates the results from both the BHK and HepG2 cell assays toasses fatty acid covalent attachment in N-terminal extension andmutation of glycosylation sites. ND=not determined.

TABLE 13 EC₅₀ values for key derivatives and hIL-22 in BHK and HepG2cell assays BHK cells HepG2 cells ID EC₅₀ (nM) EC₅₀ (nM) hIL-22 0.073.88 Derivative 1 0.48 10.13 Derivative 2 ND 6.98 Derivative 4 0.18 NDDerivative 6 0.61 14.86

Derivative 6 differs from Derivative 1 by the additional N35Q and N64Qsubstitutions (two out of three glycosylation sites mutated), yet theyare equipotent (with a tendency to slightly lower potency for Derivative6).

Whilst Derivatives 2 and 4 have a 15-mer N-terminal extension, with theCys residue for fatty acid attachment in the extension (-7C), this issurprisingly shown to be well-tolerated.

Conclusion

The potency reduction observed with fatty acid covalent attachment inthe tested derivatives of the invention was primarily driven by albuminbinding, with backbone substitutions having little contribution. Thiswas demonstrated by the surprising equipotency of Comparator 4 andhIL-22. In comparison, and as aforementioned, Genentech reports a34-fold reduction in in vitro potency for its Fc fusion of IL-22.

In the HepG2 cell assay with very low albumin levels, Derivative 1 (thederivative of IL-22 that showed a seven-fold potency reduction in theBHK assay (with albumin binding)) showed only a 2.5-fold reduction inpotency compared to hIL-22.

The equipotency of Derivatives 1 and 6 (Table 13) showed that the 35Qand 64Q mutations are surprisingly tolerated without effect on potency.

Thus, derivatives of IL-22 maintain high potency in the presence ofalbumin and are near equipotent with hIL-22 in the absence of albumin.Cys substitution and fatty acid covalent attachment are tolerated inseveral positions.

The data hence show that the derivatives of the invention demonstrategood bioavailability and potency, so offering a new and improvedtreatment for a diverse range of indications, including metabolic,liver, pulmonary, gut, kidney and skin diseases, disorders andconditions.

Example 3—In Vivo Efficacy Study in Diabetes

This study was designed to investigate the effect of once-daily dosingwith a derivative of the invention for 8-16 days in a diabetes mousemodel. The study was done in treatment (not preventive) mode, meaningthat diabetes pathology was developed before dosing was initiated. Asthe mouse model has a fatty liver (leptin receptor knockout), it alsofunctions as a metabolic model of liver disease.

Methods 7-8 week old male C57BKS db/db mice were obtained from CharlesRiver Laboratories (Day −10) and acclimatised for at least one weekprior to the start of experiments. One week after arrival (Day −3), themice were randomised and housed in groups of 10 (or singly for the foodintake study). On Day −3, and on each of Days 1-16 of the study, bloodglucose and food intake were measured.

A derivative of IL-22 (Derivative 1) was tested alongside an Fc fusionof IL-22 (hFc-hIL-22) as a comparator and vehicle only as a negativecontrol. Each agent was administered subcutaneously in a once-daily doseof 0.1, 0.25, 0.5 or 1.0 mg/kg on each of Days 1-16 days in the diabeticdb/db mice (n=6-10 in each group). Food intake was reduced followingderivative/comparator/control dosing.

Blood glucose was measured daily over the study duration. Eye bloodsamples were taken at termination in anaesthetised mice. 500 μl bloodwas collected into EDTA tubes. The samples were kept on ice andcentrifuged for five minutes at 6000 G at 4° C. within 20 minutes.Plasma was separated into 0.75 ml micronic tubes and immediately frozenfor later measurement of component concentrations.

As well as measuring derivative or comparator concentration, the plasmalevels of target engagement biomarkers (the liver-derived acute phaseproteins, haptoglobin and Serum Amyloid P component (SAP), and thegut-derived Peptide YY (PYY)) were measured at study end. Haptoglobinwas measured on a COBAS instrument (Roche Diagnostics) with commercialkit according to the manufacturer's instructions. PYY was measured witha commercial ELISA assay (ALPCO) recognising mouse and rat PYY accordingto manufacturer's instructions. SAP was measured with a commercial ELISAassay (R&D Systems) recognising mouse Pentraxin 2/SAP according tomanufacturer's instructions.

Results

Blood glucose levels over the duration study are shown in FIGS. 5 and6A.

As can be seen from FIG. 5 , hIL-22 and an hIL-22 variant havingbackbone variations only (Comparator 3) failed to reduce blood glucoseover the course of the study compared to the vehicle control.

As can be seen from FIG. 6A, Derivative 1 and hFc-hIL-22 both reducedblood glucose in a comparable manner toward normal levels with aslightly higher efficacy of Derivative 1 in the last days of the study,despite higher target engagement of hFc-hIL-22 reflecting a highersteady state exposure level in the specific study. A reduction in foodintake was observed in the treated animals compared to the vehiclecontrol (see FIG. 6B). The tested derivative thus normalised bloodglucose in the db/db model in a similar manner to hFc-hIL-22; as above,no such effect was observed with hIL-22 or Comparator 3.

The level of the target engagement biomarkers, haptoglobin, SAP and PYY,as measured at the study end, are shown in FIG. 7A-C, respectively. Ascan be seen in the graphs, all three target engagement biomarkers wereupregulated by the tested derivative and hFc-hIL-22, moreso byhFc-hIL-22 than Derivative 1.

FIG. 8 shows dose-response data for Derivative 6 (another derivative ofthe invention, it being the same as Derivative 1 but for additionalsubstitutions in two glycosylation sites). All three doses tested (0.1,0.25 and 0.5 m/kg) were effective at reducing blood glucose over time,and progressively moreso with increasing concentration.

Conclusion

Both of the tested derivatives and hFc-hIL-22 normalised blood glucosein the db/db model, thereby demonstrating an in vivo therapeutic effect.Importantly, no such effect was seen with dosing of hIL-22,demonstrating that the chronic exposure obtained with the long-actingderivatives and Fc fusion is necessary for the therapeutic effect.Whilst the mode of action for the anti-diabetic effect is not yet fullyelucidated, it is believed that IL-22 effects on the liver (hepaticgluconeogenesis and lipogenesis) are a major contributor.

Food intake was also shown to be reduced by treatment with a derivativeof the invention, thus demonstrating efficacy as an obesity treatment.

Target engagement biomarkers were also observed to be upregulated by thederivatives and hFc-hIL-22. The particular biomarkers measured in thedb/db mice are known to translate to man.

It is important to note that the circulating half-life (T_(1/2)) ofsubcutaneously administered hFc-hIL-22 is higher than for Derivative 1,specifically in mice (T_(1/2) of 20 and 8 hours, respectively; see Table7). Therefore, the exposure of hFc-hIL-22 is higher at steady state.This is further corroborated by the observation that the targetengagement biomarkers (haptoglobin, SAP and PYY) were higher in thehFc-hIL-22 group than the Derivative 1 group (FIG. 7 ) demonstratinghigher target engagement per se in the shown experiment. Thus, despitehigher exposure and target engagement, the efficacy of the Fc fusion(hFc-hIL-22) was inferior, compared to the derivative of the invention(Derivative 1), in the last three days of a 16-day dosing study.

The data hence show that the derivatives of the invention demonstrategood therapeutic efficacy in a mouse model of diabetes and liverdisease. As the particular biomarkers measured in the db/db mice areknown to translate to man, it is reasonable to predict that suchtherapeutic efficacy translates too.

Example 4—In Vivo Efficacy Study in Liver Injury (i)

This study was designed to investigate the effect of dosing withderivatives of the invention in a liver injury mouse model. The studywas done in preventive mode, meaning that liver injury was only inducedafter dosing had been initiated.

Methods

10 week-old C57B1/6 Rj mice were obtained and acclimatised for one weekbefore study start. Liver injury was induced with a singleintraperitoneal dose (300 mg/kg, 20 ml/kg) of APAP. Test derivatives ofIL-22 (Derivatives 1 and 6) were dosed at 1.5 mg/kg subcutaneously twohours prior to APAP dosing, alongside vehicle controls (n =5-10). Thestudy was terminated 24 hours after APAP dosing. A terminal bleed wassecured for measurement of plasma alanine transaminase (ALT) andaspartate transaminase (AST).

Blood samples were collected in heparinised tubes and plasma wasseparated and stored at −80° C. until analysis. ALT and AST weremeasured using commercial kits (Roche Diagnostics) on the COBAS c501autoanalyser according to the manufacturer's instructions.

Livers were subjected to formalin fixation and paraffin embedding forhistological analysis.

Proliferation was measured via ki67 immunohistochemistry (IHC) staining.IHC-positive staining was quantified by image analysis using VISsoftware (Visiopharm, Denmark).

Apoptosis was measured in a terminal deoxynucleotidyl transferase dUTPnick end labeling (TUNEL) assay. In brief, slides with paraffin-embeddedsections were de-paraffinated in xylene and rehydrated in series ofgraded ethanol. The slides were pretreated with proteinase K andendogenous peroxidase activity was blocked with hydrogen peroxide. TheTUNEL mixture (In Situ Cell Death Detection Kit, POD, Roche) was addedto the slides, followed by amplification with horseradish peroxidase(HRP) and visualization by diaminobenzidine (DAB) (Chromogen). Finally,the slides were counterstained in hematoxylin and cover-slipped.

Results

Plasma levels of ALT and AST at the termination of the study are shownin FIGS. 9A and 9B, respectively. The amount of ALT and AST was shown tobe significantly reduced in mice treated with Derivative 1 or 6 prior toliver injury compared to the vehicle/APAP control.

The number of TUNEL- and ki67-positive cells at the termination of thestudy are shown in FIGS. 10A and 10B, respectively. The amount ofTUNEL-positive cells was (significantly) reduced in mice treated withDerivative 1 or 6 prior to liver injury compared to the vehicle/APAPcontrol. The amount of ki67-positive cells was comparable across theAPAP-treated groups.

Conclusion

ALT and AST are liver enzymes used as indicators of liver damage.Derivatives 1 and 6 were hence shown to protect the liver against injuryinduced by APAP.

The results of the TUNEL assay showed that Derivatives 1 and 6 protectedagainst apoptosis caused by liver injury compared to the vehicle/APAPcontrol. Cellular proliferation, however, was unaffected by thesederivatives of IL-22. As proliferation is upregulated physiologically asa response to injury (as seen in control), the results demonstrate theproliferative action of Derivatives 1 and 6 as reduced injury is notfollowed by reduced proliferation (the ratio of proliferation overinjury is increased).

The data hence show that the derivatives of the invention demonstrategood efficacy in protecting against liver injury in a mouse model. Theparticular biomarkers measured in the mice are known to translate toman, hence it is reasonable to predict that the observed protectionwould translate too.

Example 5—In Vivo Efficacy Study in Lung Injury

This study was designed to investigate the effect of dosing with aderivative of the invention in a lung injury rat model. The study wasdone in both preventive and treatment mode, meaning that dosing wasinitiated before the lung injury was induced and continued thereafter.

Methods

To induce lung injury, 100 μl of bleomycin was administered to the lungsof male Sprague Dawley rats by oropharyngeal aspiration as a single doseon Day 1 (Groups 2 to 6). Saline was administered as a negative control(Group 1).

Animals in Groups 3, 4 and 5 were dosed (by subcutaneous injection) oncedaily with Derivative 6 at 0.5, 1.5 or 4.5 mg/kg respectively, from Day−1 to Day 3. Animals in Group 6 were dosed (by oral gavage) once dailywith prednisolone at 10 mg/kg from Day −1 to Day 3.

In order to measure soluble collagen in bronchoalveolar lavage fluid(BALF) from the rats, lungs were lavaged (3×4 ml) with sterile PBS(without calcium and magnesium) including added protease inhibitorcocktail, and the lavages per animal placed into one tube. Solublecollagen was measured in BALF supernatant using Soluble Collagen AssaySircol S1000 (Biocolor) (Charles River Laboratories).

All animals were submitted for necropsy on Day 4 (terminal euthanasia).The right lung was collected for histopathological examination from allanimals and inflation-fixed with 10% neutral buffered formalin (NBF)before being immersion-fixed in NBF. Three parallel longitudinalsections were trimmed from the right caudal lung lobe and mounted incassette 01. The right cranial, middle and accessory lung lobes werealso sectioned longitudinally and mounted in cassette 02.

Two slides were made from each cassette; one slide was stained withhaematoxylin and eosin (H&E), while the other was stained withhaematoxylin and picrosirius red (H&PSR).

Each slide was then assigned a random number using a random numbergenerator. The identification key was recorded in a Microsoft Excelspreadsheet and a copy was provided to the study pathologist followingslide evaluation. The six sections per lung were therefore read blind.

A veterinary pathologist then scored each section on each H&E-stainedslide for severity of inflammation (where 0=absent, 1=minimal, 2=mild,3=moderate and 4=severe). The mean and median score per Group wascalculated. The pathologist also scored each section on eachH&PSR-stained slide for severity of fibrosis (using a modified Ashcroftscore from to 8=high). The mean and median score per Group wascalculated and subjected to non-parametric ANOVA, Kruskal-Wallispost-test analysis.

Results

A summary of the microscopic findings is shown in Table 14, whichreveals the mean and median scores for inflammation and fibrosis foreach Group.

TABLE 14 Summary of Microscopic Findings in a Lung Injury Rat Model 3Bleo/ 4 Bleo/ 5 Bleo/ Group 1 Derivative Derivative Derivative Fibrosissaline/ 6 6 6 6 induced vehicle 2 Bleo/vehicle 0.5 mg/kg 1.5 mg/kg 4.5mg/kg Bleo/Pred No. Animals Saline Bleomycin Examined 10 10 10 10 10 10Inflammation Group 0.1 1.6 1.3 1.3 1.0 1.1 Mean Group 0.0 2.0 1.0 1.01.0 1.0 Median Fibrosis Group 0.0 1.2 0.8 0.9 0.7 0.9 Mean Group 0.0 1.01.0 1.0 0.0 1.0 Median

As evidenced by comparing Groups 1 and 2 in Table 14, bleomycin inducedboth lung inflammation and fibrosis in the rat model. The mean andmedian scores were lower in Groups 3-5, i.e. rats treated withDerivative 6, a derivative of the invention. These lower scores werecomparable with those seen in rats treated with prednisolone (Group 6).

Median inflammation and fibrosis scores for each animal in the study arealso shown in FIG. 11A and 11B, respectively.

As illustrated in FIG. 11A, the Group median inflammation score wasincreased in the bleomycin/vehicle controls (Group 2) compared withnegative controls (Group 1). The Group median inflammation scores weredecreased in rats treated with Derivative 6 (and significantly decreasedin high dose Group 5) and prednisolone (Group 6) compared withbleomycin/vehicle controls.

As illustrated in FIG. 11B, the Group median fibrosis score wasincreased in the bleomycin/vehicle controls (Group 2) compared withnegative controls (Group 1). However, Group median fibrosis scores weredecreased in rats treated with Derivative 6 (and significantly decreasedin high dose Group 5) compared with bleomycin/vehicle controls but notwith the control prednisolone.

As illustrated in FIG. 11C, the amount of soluble collagen in BALF afterbleomycin-induced lung injury was increased in the bleomycin/vehiclecontrols (Group 2) compared with negative controls (Group 1), and thiswas not reduced by treatment with prednisolone (Group 6). However, asignificantly reduced amount of soluble collagen was observed in BALFfrom rats treated with Derivative 6 (across all doses tested) comparedwith bleomycin/vehicle controls. As soluble collagen in BALF is aread-out for fibrosis, these results confirm the histology data reportedimmediately above.

Conclusion

The results of the microscopic studies showed that the derivatives ofthe invention are able to prevent and/or reduce bleomycin-induced lunginflammation and fibrosis in a rat model. The effects seen with respectto inflammation were comparable to those observed with prednisolone, acorticosteroid known for treating lung inflammation. However, thederivatives of the invention had a unique action on fibrosis, not seenwith prednisolone.

Example 6—In Vivo Efficacy Study in Colitis

This study was designed to investigate the effect of dosing with aderivative of the invention in a colitis mouse model. The study was donein both preventive and treatment mode, meaning that dosing was initiatedon the same day as the colon inflammation was induced and continuedthereafter.

Methods

Chow-fed female C57B 1/6 JRj mice were randomised to five groups (n=8per group) based on body weight. DSS was used to induce colitis in fourof the five groups. These mice received DSS in their drinking water forseven days from study day 0 to 6. In the fifth group, animals receivedwater without DSS and hence served as healthy controls. From study dayDSS mice were treated with vehicle, a test derivative of IL-22(Derivative 6; at 0.35 mg/kg or 1 mg/kg dosed intraperitoneally) or anIL-22-Fc fusion as comparator (hFc-hIL-22; at 0.5 mg/kg dosedintraperitoneally) once daily for 10 days. Body weight, food and waterintake were monitored daily.

On study day 10, blood samples were collected from the mice in EDTAtubes and the plasma was separated and stored at −80° C. until analysis.Regenerating Islet Derived Protein 3 Gamma (Reg3g) was measured induplicates using an ELISA kit (Cloud-Clone Corp), according to themanufacturer's instructions. Reg3g is a target engagement marker ofIL-22.

At termination, the intestines were removed for stereological analysis.Accordingly, the gut was flushed with ice cold saline and its contentgently removed before sampling.

The intestine was infiltrated in formalin overnight (Tissue-Tek VIP) andsubsequently embedded in blocks of paraffin. The formalin-fixedintestine was then sampled from the proximal to the distal directionusing systematic uniform random sampling (SURS) principles, resulting ina total of four slabs and placed in a multi-cassette. All tissue slabswere placed in such a way that identification of individual slabs waspossible at a later stage.

The paraffin blocks were trimmed and 5 μm top sections were cut andmounted on Superfrost+ object glasses. For the large intestine, anothersection was cut with a 500 μm distance to the top section, thus givingrise to a total of eight colon sections from each animal.

Colon inflammation volume was measured stereologically, i.e. using athree-dimensional interpretation of two-dimensional cross sections ofthe colon. The stereological volume estimation was performed using thenewCAST system (Visiopharm) on scanned H&E-stained slides. Total gutvolume, volume of mucosa, volume of submucosa and muscularis and volumeof inflamed tissue were estimated by point counting using a grid systemof appropriate size, where all points hitting the structure of interestwere counted. The number of points hitting the structure of interestwere converted into volume according to the following mathematicalrelationship:

Vol_(ref)=Σp·A(p)·t

where A(p) is the area per point, p is the total number of pointshitting the structure of interest and t is the distance betweensections. The mean inflammation volume per group was calculated andsubjected to statistical analysis.

Colon morphology was also assessed at termination by viewing theH&E-stained slides.

Results

Colon inflammation volume is shown in FIG. 12 . Inflammation was shownto be prevented in mice treated with Derivative 6, at either dose,compared to the vehicle control (also containing DSS). Notably,inflammation remained at normal levels in the groups treated withDerivative 6, as evidenced by the colon inflammation volume being thesame for the treated groups as the healthy controls (vehicle with noDSS). The same was true for the group treated with hFc-hIL-22.

Representative H&E staining images of colon morphology at terminationare shown in FIG. 13 . Following DSS treatment, mucosal epithelialwounding can be seen in vehicle-treated animals (marked by black arrow),but not in animals treated with Derivative 6 at either dose orhFc-hIL-22. This demonstrates a protective effect on epithelial tissue.

Plasma Reg3g levels are shown in FIG. 14 . DSS treatment induced anincrease in basal Reg3g levels (compare vehicle to no DSS vehicle). Nofurther increase was detectable in the low dose (0.35 mg/kg) Derivative6 group, but was seen in the higher dose (1 mg/kg) Derivative 6 groupand the hFc-hIL-22 group. The higher Reg3g levels in the hFc-hIL-22 (0.5mg/kg) group compared to the Derivative 6 (1 mg/kg) group indicatedhigher target engagement despite the lower dose, which was likelyrelated to the longer half-life in mice of hFc-hIL-22 (T½ of 30 hoursfor hFc-hIL-22 vs 9.1 hours for Derivative 6).

Conclusion

The data hence show that a derivative of the invention demonstrates goodefficacy in protecting against colitis and mucosal epithelial woundingin a mouse model. This indicates that a new and improved treatment forgut diseases, disorders and conditions has been found. In particular,the findings demonstrate the potential to treat disease characterised bymucosal epithelial damage, such as inflammatory bowel disease.

Example 7—In Vivo Efficacy Study in Liver Injury (ii)

This study was designed to investigate the effect of dosing with aderivative of the invention in a second liver injury mouse model (thefirst being described above in Example 4). The study was done inpreventive mode, meaning that liver injury was only induced after dosinghad been initiated.

Methods

C57B16/6j male mice were divided into five groups (n=8 per group). Atest derivative of IL-22 (Derivative 1) was dosed at 1 mg/kgintraperitoneally in two of the five groups at −26 hours and −2 hoursrelative to ConA treatment. Another two groups received vehicle only atthese time points. ConA was given to all four groups as an intravenousbolus over a 30-second period at a dose of 15 mg/kg, to induce liverinjury. The fifth group received no ConA (vehicle only, as above), ashealthy controls.

8 or 24 hours after ConA injection, the mice were placed underisoflurane anaesthesia and the maximal volume of blood was taken bycardiac puncture (using a polypropylene serum gel tube containing a clotactivator). Mice receiving no treatment (Group 5) were sacrificed at the8-hour time point. The blood was mixed with the clotting activationagent in each tube by inverting the tube several times. The tube wasmaintained for 15 minutes at room temperature and then centrifuged at2000 g for 10 minutes at 4° C. ALT and AST were measured in the serumsamples using an automated system (Konelab 20) according tomanufacturer's instructions.

Results

Plasma levels of ALT and AST at the termination of the study are shownin FIGS. 15A and 15B, respectively. The amount of ALT and AST was shownto be reduced in mice treated with Derivative 1 prior to liver injurycompared to the vehicle/ConA control, at both time points tested.

Conclusion

ALT and AST are liver enzymes used as indicators of liver damage.Derivative 1 was hence shown to protect the liver against injury inducedby ConA, just as it had against injury induced by APAP in Example 4. Theparticular biomarkers measured in the mice are known to translate toman, hence it is reasonable to predict that the observed protectionwould translate too.

Example 8—In Vivo Efficacy Study in Obesity and NASH

This study was designed to investigate the effect of dosing with aderivative of the invention in an obese and NASH mouse model. The studywas done in treatment (not preventive) mode, meaning that obesity andNASH pathology was developed before dosing was initiated.

Methods

The Diet Induced Obese mouse model was based on male C57BL/6JRj mice,which were fed a high fat diet for at least 30 weeks prior to theexperiment. The diet was high in fat (40%), fructose (22%) andcholesterol (2%) (Research Diets D09100310). This resulted in obesity,NAFLD and ultimately NASH.

The animals were single-housed six days prior to the first dose of testderivative (or other) and body weight was monitored daily throughout theexperiment. The mice were divided into six groups (n=12 per group).Dosing was initiated at study day 0 (indicated with dotted line in FIG.16 ) and administered once daily subcutaneously in the following doses.

Semaglutide, a long acting GLP-1 receptor agonist, was used a positivecontrol in a first group, and also investigated in combination with atest derivative of IL-22 (Derivative 6) in a second group. The dosing ofsemaglutide was gradually titrated according to the following schedule:0.6 nmol/kg day 0-1.2 nmol/kg day 1-2.4 nmol/kg day 2-4.8 nmol/kg day3-12 nmol/kg day 4-30 nmol/kg day 5. In the combination group,semaglutide dosing was initiated at day 0 and Derivative 6 dosing wasinitiated at day 12 (indicated with dotted line in FIG. 16 ) afterweight loss had plateaued in the semaglutide treated group.

The dosing of Derivative 6 in a third, “high dose” group was graduallytitrated according to the following schedule: 0.05 mg/kg day 0-0.1 mg/kgday 1-0.15 mg/kg day 2-0.2 mg/kg day 3-0.25 mg/kg day 4. The dose wasswitched from 0.25 mg/kg to 0.1 mg/kg at day 14 (indicated with dottedline in FIG. 16 ). In a fourth, “low dose” group, dosing of Derivative 6started at 0.05 mg/kg with no further titration.

In a fifth group, the dosing of an IL-22-Fc fusion (hFc-hIL-22) ascomparator was gradually titrated according to the following schedule:0.02 mg/kg day 0-0.04 mg/kg day 1-0.06 mg/kg day 2 — 0.08 mg/kg day3-0.1 mg/kg day 4. The dose of hFc-hIL-22 was chosen to match the 0.25mg/kg Derivative 6 group for target engagement based on the longerhalf-life and correspondingly higher target engagement compared toDerivative 6 as seen in Example 6 (see FIG. 14 ).

The sixth group were dosed with vehicle only as negative controls.

Plasma triglyceride (TG) levels were measured at baseline (day −2), week2 (day 14) and week 4 (day 28) after dosing initiation. Specifically,tail blood samples were collected for analysis by pressing a tail bloodvolume at or below 200 μl into an open Microvette (100 μl or 200 μl)tube treated with the appropriate anticoagulant. Blood was placed at 4°C. until it was centrifuged at 3000 g for 10 minutes. The plasmasupernatants were transferred to new tubes and immediately frozen on dryice and stored at −80° C. TG levels were measured using commercial kits(Roche Diagnostics) on the cobas® c 501 autoanalyzer according to themanufacturer's instructions.

Results

Body weight over the course of the experiment is illustrated in FIG. 16.

The study demonstrated dose-dependent high efficacy of Derivative 6 inlowering body weight in the obese mouse model. Furthermore, itdemonstrated additivity to a GLP-1 receptor agonist (semaglutide), whichis being investigated in late stage clinical trials for obesitytreatment. The data suggested superiority of Derivative 6 compared tohFc-hIL-22 in inducing weight loss. Importantly, hFc-hIL-22 has a longerhalf-life in mice than Derivative 6, and has demonstrated higher targetengagement even when dosed at half the dose of Derivative 6. Thus, thedose of 0.1 mg/kg hFc-hIL-22 used in this study was chosen for similartarget engagement as the 0.25 mg/kg Derivative 6 group.

The sensitivity to weight loss induced by Derivative 6, here observed inDiet Induced Obese mice, is not observed in lean mice. For example, in a10-day DSS-induced colitis study (Example 6) with once daily dosing,body weight at study initiation was 19.0 g in both the DSS/vehicle groupand the DSS/Derivative 6 (0.35 mg/kg) group. At the end of the study thebody weight was 17.6 gram in the DSS/vehicle group versus 17.4 gram inthe DSS/Derivative 6 (0.35 mg/kg) group, which are not different (p=0.82in unpaired students t-test). By comparison, mice in the vehicle andDerivative 6 (0.25 mg/kg) groups at day 10 in the present study had bodyweights of 43.5 g and 35.2 g, respectively. Thus, a significant weightloss was observed in the Derivative 6 group compared to the vehiclegroup (p<0.0001 in unpaired students t-test for body weight at day 10).At study initiation, the body weight was similar in the vehicle groupversus the Derivative 6 group (44.3 g and 44.1 g, respectively).

Plasma TG levels, as measured at baseline (day −2), week 2 (day 14) andweek 4 (day 28) following dosing initiation, are shown in Table 15.

TABLE 15 Plasma TG levels (nmol/l) in an obese and NASH mouse modelSemaglutide Derivative 6 (30 nmol/kg) + Derivative 6 (0.25/0.1Semaglutide Derivative 6 Vehicle hFc-hIL-22 (0.05 mg/kg) mg/kg) (30nmol/kg) (0.05 mg/kg) Mean SEM Mean SEM Mean SEM Mean SEM Mean SEM MeanSEM Baseline 0.75 0.033 0.85 0.036 0.83 0.045 0.77 0.035 0.81 0.036 0.860.046 Week 2 1.00 0.059 0.68 0.043 0.58 0.038 0.52 0.027 0.65 0.037 0.460.033 Delta +0.24 0.071 −0.18 0.047 −0.25 0.050 −0.25 0.050 −0.15 0.046−0.41 0.066 week 2 Week 4 0.93 0.076 0.84 0.046 0.54 0.030 0.42 0.0300.66 0.048 0.34 0.032 Delta +0.17 0.079 −0.02 0.059 −0.30 0.034 −0.350.052 −0.14 0.066 −0.53 0.042 week 4

Delta refers to the change in TG level (nmol/1) from baseline for theindicated treatment and timepoint. As can be seen from Table 15, thechange in level was positive (increased) for the vehicle group, butnegative (decreased) for all other groups.

Derivative 6 had higher efficacy in lowering TG levels than semaglutide,also in the low dose, which causes less weight loss than semaglutide(e.g. week 4 delta TG levels (nmol/l) of −0.14±0.066 for semaglutide,−0.30±0.034 for Derivative 6 (0.05 mg/kg) and −0.35±0.052 for Derivative6 (0.25/0.1 mg/kg)). The results show that Derivative 6 had a highefficacy in TG lowering, which is partially independent of the weightloss effect. Furthermore, there was full additivity of the Derivative 6effect on top of semaglutide. At week 4, the lowering of TG levels(nmol/l), calculated as delta TG, was −0.30±0.034 for Derivative 6 (0.05mg/kg), −0.14±0.066 for semaglutide and −0.53±0.042 for semaglutide+Derivative 6 (0.05 mg/kg). The TG lowering results compared to baseline(delta TG) were despite TG levels in the vehicle group increasing overthe course of the study.

Conclusion

The study demonstrated that a derivative of the invention (Derivative 6)can induce weight loss in obese mice in a dose-dependent manner at leastto a comparable level as seen with semaglutide—a long acting GLP-1receptor agonist used a positive control. Furthermore, there was anadditive effect on weight loss using a combination of semaglutide andDerivative 6. The efficacy of Derivative 6 in inducing weight loss washigher than observed with hFc-IL-h22 at doses chosen to give a similarlevel of target engagement. The weight loss induced by Derivative 6 inDiet Induced Obese mice was not seen in DSS-treated lean mice,demonstrating that obese mice are more sensitive to Derivative 6-inducedweight loss. As body weight loss observed in Diet Induced Obese mice isthe same read-out as would be used in man, it is reasonable to predictthat the observed weight loss would translate too.

Derivative 6 also showed high efficacy in lowering TG levels. Derivative6, in both doses tested, showed higher efficacy than semaglutide andfull additivity on efficacy in combination dosing was observed. Giventhat the 0.05 mg/kg dose of Derivative 6 had higher efficacy thansemaglutide despite lower weight loss, it can be concluded that the TGlowering effect of Derivative 6 was at least partially independent ofweight loss. Furthermore, Derivative 6 was superior to the hFc-hIL-22comparator at both of the tested doses. As the TG lowering observed inDiet Induced Obese mice was the same read-out as would be used in man,it is reasonable to predict that the observed effect would translatetoo. The results hence indicate that a new treatment for disorders andconditions characterised by high TG levels has been found.

While certain features of the invention have been illustrated anddescribed herein, many modifications and equivalents will occur to thoseof ordinary skill in the art. It is, therefore, to be understood thatthe claims are intended to cover all such modifications and equivalentsas fall within the true spirit of the invention.

1. A method of treating a disease, disorder, or condition in a subject,wherein the method comprises administering an effective amount of aderivative of IL-22 to the subject, and wherein the derivative of IL-22.comprises a fatty acid covalently attached to an IL-22 protein, wherein:(i) the IL-22 protein is native mature human IL-22 (hIL-22; SEQ IDNO. 1) or a variant thereof, wherein the variant (a) comprises avariation within SEQ ID NO. 1 and has at least 10% sequence identitywith hIL-22, and/or (b) comprises a variation relative to SEQ ID NO. 1;and (ii) the fatty acid is covalently attached to a Cys residue in theIL-22 protein.
 2. The method of claim 1, wherein the fatty acid iscovalently ched to the Cys residue by a linker.
 3. The method of claim1, wherein the disease, disorder or condition is a metabolic, liver ;pulmonary, gut, kidney or skin disease, disorder or condition.
 4. Themethod of claim 3, wherein: (i) the metabolic disease, disorder orcondition is obesity, diabetes type I, diabetes type 2, hyperlipidemia,hyperglycemia or hyperinsulinemia; (ii) the liver disease, disorder orcondition is non-alcoholic fatty liver disease (NAELD), non-alcoholicsteatohepatitis (NASH), cirrhosis, alcoholic hepatitis, acute liverfailure, chronic liver failure, acute-on-chronic liver failure (ACLF),acetaminophen induced liver toxicity, acute liver injury, sclerosingcholangitis, biliary cirrhosis or a pathological condition caused bysurgery or transplantation; (iii) the pulmonary disease, disorder orcondition is chronic obstructive pulmonary disease (COPD), cysticfibrosis, bronchiectasis, idiopathic pulmonary fibrosis, acuterespiratory distress syndrome, a chemical injury, a viral infection, abacterial infection or a fungal infection; (iv) the gut disease,disorder or condition is inflammatory bowel disease (IBD), ulcerativecolitis, Crohn's disease, graft- versus-host-disease (GVHD), a chemicalinjury, a viral infection or a bacterial infection; (v) the kidneydisease, disorder or condition is acute kidney disease or chronic kidneydisease; or (vi) the skin disease, disorder or condition is a wound,inflammatorydisease or GvHD.
 5. The method of claim 1, wherein themethod comprises administering a daily dose of between 0.001 μg/kg ofbody weight and 10 mg/kg of body weight of the derivative to thesubject.
 6. The method of claim 2, wherein (i) the variant has thesequence set forth in SEQ ID NO. 16, the linker is γGlu-OEG-OEG-C₂DA-Ac,the fatty acid is a C18 diacid and the linker is attached to the Cysresidue substituted at position 1 of hIL-22 (identified herein asDerivative 1); (ii) the variant has the sequence set forth in SEQ ID NO.17, the linker is γGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C18 diacidand the linker is attached to the Cys residue at position −7 relative tohIL-22 (identified herein as Derivative 2); (iii) the variant has thesequence set forth in SEQ ID NO. 16, the linker is γGlu-OEG-OEG-C₂DA-Ac,the fatty acid is a C16 diacid and the linker is attached to the Cysresidue substituted at position 1 of hIL-22 (identified herein asDerivative 3); (iv) the variant has the sequence set forth in SEQ ID NO.17, the linker is γGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C16 diacidand the linker is attached to the Cys residue at position −7 relative tohIL-22 (identified herein as Derivative 4); (v) the variant has thesequence set forth in SEQ ID NO. 16, the linker isγGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc, the fatty acid is a C14 diacid andthe linker is attached to the Cys residue substituted at position 1 ofhIL-22 (identified herein as Derivative 5); (vi) the variant has thesequence set forth in SEQ ID NO. 18, the linker is γGlu-OEG-OEG-C₂DA-Ac,the fatty acid is a C18 diacid and the linker is attached to the Cysresidue substituted at position 1 of hIL-22 (identified herein asDerivative 6); (vii) the variant has the sequence set forth in SEQ IDNO. 19, the linker is γGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C18diacid and the linker is attached to the Cys residue substituted atposition 1 of hIL-22 (identified herein as Derivative 7); (viii) thevariant has the sequence set forth in SEQ ID NO. 20, the linker isγGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C18 diacid and the linker isattached to the Cys residue substituted at position 6 of hIL-22(identified herein as Derivative 8); (ix) the variant has the sequenceset forth in SEQ ID NO. 21, the linker is γGlu-OEG-OEG-C₂DA-Ac, thefatty acid is a C18 diacid and the linker is attached to the Cys residuesubstituted at position 33 of hIL-22 (identified herein as Derivative9); or (x) the variant has the sequence set forth in SEQ ID NO. 18, thelinker is γGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C16 diacid and thelinker is attached to the Cys residue substituted at position 1 ofhIL-22 (identified herein as Derivative 10).
 7. A method of treating adisease, disorder, or condition in a subject, wherein the methodcomprises administering an effective amount of a pharmaceuticalcomposition to the subject, wherein the pharmaceutical compositioncomprises a derivative of IL-22, and wherein the derivative of IL-22comprises a fatty acid coxal eptly attached to an IL-22 protein,wherein: (i) the IL-22 protein is native mature human IL-22 (hIL-22; SEQID NO. 1) or a variant thereof, wherein the variant (a) comprises avariation within SEQ ID NO. 1 and has at least 10% sequence identitywith hIL-22, and/or (b) comprises a variation relative to SEQ ID NO. 1;and (ii) the fatty acid is covalently attached to a Cys residue in theIL-22 protein.
 8. The method of claim 7, wherein the fatty acid iscovalently attached to the Cys residue by a linker.
 9. The method ofclaim 8, wherein (i) the variant has the sequence set forth in SEQ IDNO. 16, the linker is γGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C18diacid and the linker is attached to the Cys residue substituted atposition 1 of hIL-22 (identified herein as Derivative 1); (ii) thevariant has the sequence set forth in SEQ ID NO. 17, the linker isγGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C18 diacid and the linker isattached to the Cys residue at position -7 relative to hIL-22(identified herein as Derivative 2); (iii) the variant has the sequenceset forth in SEQ ID NO. 16, the linker is γGlu-OEG-OEG-C₂DA-Ac, thefatty acid is a C16 diacid and the linker is attached to the Cys residuesubstituted at position 1 of hIL-22 (identified herein as Derivative 3);(iv) the variant has the sequence set forth in SEQ ID NO. 17, the linkeris γGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C16 diacid and the linkeris attached to the Cys residue at position −7 relative to hIL-22(identified herein as Derivative 4); (v) the variant has the sequenceset forth in SEQ ID NO. 16, the linker isγGlu-γGlu-γGlu-γGlu-OEG-OEG-εLys-αAc, the fatty acid is a C14 diacid andthe linker is attached to the Cys residue substituted at position 1 ofhIL-22 (identified herein as Derivative 5); (vi) the variant has thesequence set forth in SEQ ID NO. 18, the linker is γGlu-OEG-OEG-C2DA-Ac,the fatty acid is a C18 diacid and the linker is attached to the Cysresidue substituted at position 1 of hIL-22 (identified herein asDerivative 6); (vii) the variant has the sequence set forth in SEQ IDNO. 19, the linker is γGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C18diacid and the linker is attached to the Cys residue substituted atposition 1 of hIL-22 (identified herein as Derivative 7); (viii) thevariant has the sequence set forth in SEQ ID NO. 20, the linker isγGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C18 diacid and the linker isattached to the Cys residue substituted at position 6 of hIL-22(identified herein as Derivative 8); (ix) the variant has the sequenceset forth in SEQ ID NO. 21, the linker is γGlu-OEG-OEG-C₂DA-Ac, thefatty acid is a C18 diacid and the linker is attached to the Cys residuesubstituted at position 33 of hIL-22 (identified herein as Derivative9); or (x) the variant has the sequence set forth in SEQ ID NO. 18, thelinker is γGlu-OEG-OEG-C₂DA-Ac, the fatty acid is a C16 diacid and thelinker is attached to the Cys residue substituted at position 1 ofhIL-22 (identified herein as Derivative 10).
 10. The method of claim 6,wherein the disease, disorder or condition is a metabolic, liver,pulmonary, gut, kidney or skin disease, disorder or condition.
 11. Themethod of claim 7, wherein the disease, disorder or condition is ametabolic, liver, pulmonary, gut, kidney or skin disease, disorder orcondition.
 12. The method of claim 9, wherein the disease, disorder orcondition is a metabolic, liver, pulmonary, gut, kidney or skin disease,disorder or condition.
 13. A method for preparing a derivative of IL-22,comprising covalently attaching a fatty acid to an 1L-22 protein,wherein: (i) the IL-22 protein is native mature human IL-22 (hIL-22; SEQID NO. 1) or a variant thereof, wherein the variant (a) comprises avariation within SEQ ID NO. 1 and has at least 10% sequence identitywith hIL-22, and/or (b) comprises a variation relative to SEQ ID NO. 1;and (ii) the fatty acid is covalently attached to a Cys residue in theIL-22 protein.